环境中有机磷酸酯阻燃剂(TDCP、TECP和TCPP)对人胚肾
细胞(HEK 293)的影响
任相浩;陈雅岚;寇莹莹
【摘要】水环境中的微污染物有机磷酸酯如三(2-氯乙基)磷酸酯(TCEP)、三(2-氯-异丙基)磷酸酯( TCPP)和三(1,3-二氯丙基)磷酸酯(TDCP)主要用作聚氨酯泡沫塑料的阻燃剂.物理、化学和生物处理很难完全消除这些污染物.研究阻燃剂在环境水平和较高浓度下对人细胞系的细胞毒性和细胞周期效应.结果表明,在浓度为0. 001 mg/L和0. 01 mg/L时,只有TDCP具有轻微的细胞毒性,而当这三种化学物质浓度100 mg/L以上时对细胞毒性有显著的诱导作用. TCEP和TCPP的EC50分别为276. 8 mg/L和58. 4 mg/L.三种药物均能抑制CDK 4的表达,TDCP能增加CDK 2和cyclin E的表达,而TCEP和TCPP在CDK 2和cyclin E的表达上表现出自差现象. TDCP和TCEP使细胞数量减少,细胞形态发生改变.可见三种化学物质对HEK 293细胞的杀伤作用可能是通过抑制CDK 4调节蛋白而产生的.
【期刊名称】《科学技术与工程》
【年(卷),期】2019(019)010
【总页数】7页(P254-260)
【关键词】有机磷阻燃剂;微量污染物;环境水平;细胞毒性;细胞周期调节蛋白;人体细胞
【作者】任相浩;陈雅岚;寇莹莹
【作者单位】北京建筑大学环境与能源工程学院城市雨水系统与水环境教育重点实验室,北京100044;北京建筑大学环境与能源工程学院城市雨水系统与水环境教育重点实验室,北京100044;北京建筑大学环境与能源工程学院城市雨水系统与水环境教育重点实验室,北京100044
【正文语种】中文
【中图分类】X171.5
Being a major component of chlorinated organophosphate flame retardants, tris-(1,3-dichloro-isopropyl)-phosphate (TDCP), tris-(2-chloroethyl)-phosphate (TCEP) and tris-(2-chloro-isopropyl)-phosphate (TCPP) are mostly used in polyurethane foams. These retardants as typical micropollutants present in surface water, wastewater treatment plant effluent, ocean and drinking water from ng/L to μg/L level normally called as environmental level [1—3]. The elimination of these three organophosphate retardants is 0～30% by conventional wastewater treatment plant and membrane bioreactor (MBR), about 50% by ozonization, and 83%～96% by biologically active slow sand filters and GAC filtration[3—7]. Even in reverse osmosis (RO) and nanofiltration (NF), maximum removal efficiency is 95% [6,8]. Therefore, the importance of low concentration of these fire retardants remained in aquatic environment has been increasing on human health[9].
In cytotoxic study of these organophosphate retardants, only few people have investigated toxic effects using in vitro test, especially on human cells,
whereas major studies were progressed by in vivo test[10]. Föllmann and Wober[11] studied cytotoxic, genotoxic, mutagenic and estrogenic effects of TCEP and TCPP using hamster fibroblasts (V79) cells with various concentrations from low ng/L to high mg/L, and detected slight cytotoxic effect at mg/L level. Dishaw et al[12] investigated neurotoxicity of TDCP, TCEP and TCPP using PC12 cells, and the results showed that those chemicals might affect neurodevelopment. Ren et al[13] studied cytotoxic and molecular toxic effects of TCEP on primary cultured rabbit renal proximal tubule cells, and TCEP induced cytotoxic and molecular toxic effects at environmental level. However, in spite of sensitive detection, the culture of primary animal cells was not easy to be handled in the experiments.
In this research, a typical human cell line, HEK293 cells, was used to investigate toxic effects of target chemicals conducted from waster sources, where the culture of cell lines is much easier than primary cells [14,15]. Due to investigation of environmentally typical micropollutants, both environmental concentration (0.001 mg/L and 0.01 mg/L) and higher concentrations were considered with 48 h exposure time on the
cells[13,16]. Expression of cell cycle regulatory proteins was also investigated to study mechanism of cytotoxic effect, where cell cycle regulatory proteins control eukaryotic cell growth that is composed of the gap 1 phase (G1), DNA synthesis phase (S), gap 2 phase (G2), and mitosis phase (M)[17]. Thus, the purpose in this study was to investigate cytotoxic and molecular toxic effect of TDCP, TCEP and TCPP at environmental and
higher level on the typical human cell line.
1 Materials and methods
1.1 Chemical samples and antibodies
Tris-(2-chloroethyl)-phosphate (TCEP) (CAT-No.119660-25G) was from Sigma-Aldrich (USA). Tris-(1,3-dichloro-isopropyl)-phosphate (TDCP) (CAT-No. 203-06892) and tris-(2-chloro-isopropyl)-phosphate (TCPP) (CAT-No. 200-13811) were purchased from Wako (Japan). Primary antibodies of cyclin dependent kinase 4 (CDK4) (CAT-No.sc-23896), cyclin dependent kinase 2 (Cdk2) (CAT-No.sc-6248), cyclin E (CAT-No.sc-247) and β-actin (CAT-No.sc-47778) were acquired from Santa Cruz Biotechnology (USA). Goat anti-mouse (CAT-No.sc-2005) as a secondary antibody was also obtained from Santa Cruz Biotechnology (USA).
1.2 Cell culture
Human embryonic kidney 293 cells were purchased from American Type Culture Collection (ATCC CRL-1573). The cells were cultured in Dulbecco’s modified eagle medium (DMEM) (GIBCO 11995-065, Invitrogen, USA) with addition of 10% fetal bovine serum (FBS) (GIBCO 16000-044, Invitrogen, USA). The cells were maintained in CO2 incubator with 37 ℃ and 5% CO2 concentration. In the sub-culture, HEK293 cells were washed by Dulbecco’s phosphate-buffered saline (PBS) (GIBCO 142000-075, Invitrogen, USA) solution and then harvested by trypsin (GIBCO 12604-013, Invitrogen, USA).
1.3 Sulforhodamine B (SRB) assay and cell morphological analysis
Ten thousands cells of HEK293 cells were cultivated to each well in 96-well
plate and maintained in CO2 incubator for 12 h, where DMEM culture media was used to culture the cells. These cells were then treated by the three chemicals with twelve different concentrations for 48 h. Fifty micro-liter of 50% cold TCA was added to each well slowly and kept in the refrigerator for 1 h with 4 ℃. The plate was washed by tap water, and then dried for 1 h in room temperature. One hundred micro-liter of 0.4% SRB was added to each well and dried more than 30 min. Again, the plate was washed five times by 1% acetic acid, and then dried for 2 h in room temperature. One hundred fifty micro-liter of 10 mmol/L unbuffered Tris was added to each well and detected by microplate reader with 550 nm. Also, with 48 h exposure of three chemicals to HEK293 cells, the cell morphology was detected by an inverted microscopy (Olympus IX-51) with 20×10 magnification.
1.4 Western blotting assay
HEK293 cells cultivated in 60Ø dish were washed twice with ice-cold PBS, and lysed in SDS buffer. The lysed cells were collected and moved to E-tube. The Etube was boiled at 90 ℃ for 5 min. The boiled cells were triturated 20 times by 1 mL syringe and then centrifuged at 12 000 rpm for 10 min. The supernatants were collected as a total cell fraction. The protein was quantified by Quant-iTTM protein assay (Q33211, Invigtrogen, USA). The cell homogenates (30 μg of protein) were separated on Nupage 10% Bis-Tris Gel (NP0315BOX, Invitrogen, USA) with 20× NuPA GE MOPS SDS Running Buffer (NP0001, Invitrogen, USA). The separated proteins were transferred from the gel to nitrocellulose membrane (IB3010-01, Invitrogen,
USA) by iBlot Transfer (692404, Invitrogen, USA). The membranes were then washed with water and then blocked with 5% skimmed milk powder in TBS-T (10 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 0.05% Tween-20) for 1 h. The blocked membrane were washed three times with TBS-T, and treated with primary antibody at 4 ℃ for overnight. After treatment of secondary antibody, the bands were visualized with enhanced chemiluminescence (Amersham Pharmacia Biotech, England, UK).
1.5 Statistical analysis
The results were expressed as the mean ± standard error (SE), and were also analyzed by t-test and ANOVA, where P<0.05 and P<0.01 were considered significant.
2 Results and analyese
2.1 Cytotoxic effects of three environmental chemicals on HEK293 cells Cytotoxicity of TDCP, TCEP and TCPP was observed from μg/L to mg/L level by SRB assay. TDCP slightly decreased cell viability at environmental concentrations (0.001 mg/L and 0.01 mg/L), showing 95.0% and 96.6% of the control, respectively [Fig.1(a)]. From 50 mg/L, TDCP significantly decreased cell viability, ranging in 82.2%～87.4% of the control. TCEP did change cell viability from 0.001 mg/L to 75 mg/L, compared with the control, but significantly decreased from 100 mg/L, 27.7%～94.1% of the control [Fig.1(b)]. TCPP inFig.1(c) did not affect cell viability until 1 mg/L, but significantly decreased from 50 mg/L and upper concentrations, showing 2
3.5%～71.4% of the control.
*represents for P<0.05 vs control; ** represents for P<0.01 vs controlFig.1
Cytotoxic effects of TDCP, TCEP and TCPP on HEK293 cells for 48 h exposure with different concentrations
The half effective concentration (EC50) as one expressional way of cytotoxic effects was also calculated using Origen 6.1 software based on the cell viability data shown above (Table 1). Because there was no dose dependent change in TDCP treated samples, no EC50 value was possibly evaluated. The EC50 of TCEP was 276.8 mg/L and TCPP was four times lower than TCEP, showing 58.4 mg/L.
Table 1 EC50 values of three target chemical samples for 48 h exposureEnvironmental chemicalsEC50 value in HEK293 cells/(mg·L-
1)TDCPNDTCEP276.8TCPP58.4
ND: not detected.
2.2 Morphological and numerical effect of three chemicals
Table 2 showed morphological and numerical effects of HEK293 cells on TDCP, TCEP and TCPP at three different concentrations through microscopic study with 48 h chemical exposure. In the TDCP treated cells, morphological and numerical change was not clearly found at 0.01 mg/L, but cell number was slightly decreased at 1 mg/L and 50 mg/L, and clearly decreased at 100 mg/L, compared with the control. TCEP did not affect on the morphological change of HEK293 cells from 0.01 mg/L to 100 mg/L, but slightly decreased cell number at 100 mg/L, whereas did not change cell number at 0.01 mg/L 1 mg/L and 50 mg/L, compared with the control. In the TCPP treated cells, both morphological and numerical changes were clearly found at 50 mg/L and 100 mg/L, compared with the control, and
more number of shrunken cells was found at 100 mg/L than at 50 mg/L. Not clear change of cell number was found at 0.01 mg/L and 1 mg/L, compared with the control.
2.3 Effect of three chemicals on cell cyclic regulatory protein expression
In the results of mechanism study, TDCP at 0.01 mg/L significantly inhibited the expression of CDK4, one of typical cell cycle regulatory proteins, but slightly decreased the expression at 1 mg/L and 50 mg/L, compared with the control (Fig.2). However, TDCP slightly increased the expression of CDK2 at 1 mg/L and 50 mg/L, whereas no effect was found at 0.01 mg/L, compared with the control. The expression of cyclin E was induced by 1 mg/L and 50 mg/L of TDCP, but no effect by 0.01 mg/L, compared with the control.
The expression of cell cycle regulatory proteins treated by TCEP was also introduced in Fig.3.TCEP significantly inhibited the expression of CDK4 at tested three concentrations, but slightly increased the expression of CDK2 at 1 mg/L and 50 mg/L. The expression of cyclin E was significantly induced at 0.01 mg/L, and slightly increased at 1 mg/L, however, significantly decreased at 50 mg/L, compared with the control.
Table 2 Morpholog ical analysis of HEK293 cells (20× magnification) treated with three chemicals after 48 h of exposure
β-actin was used as the internal standard; the lower panels denote the means ± standard errorFig.2 Effect of TDCP-treated HEK293 cells on cell cycle regulatory protein expression
TCPP slightly increased the expression of CDK4 at 0.01 mg/L, but significantly inhibited the expression at 1 mg/L and 50 mg/L (Fig.4. The expression of CDK2 was increased at all tested concentrations. TCPP significantly increased the expression of cyclin E at 0.01 mg/L, but significantly decreased the expression at 50 mg/L, compared with the control.
3 Discussions
In the cytotoxic results conducted from this research, TDCP slightly decreased cell viability at 0.001 mg/L and 0.01 mg/L, but significantly inhibited from 50 mog/L on HEK293 cells, and Liu et al[18] reported similar results that less than 80% cell viability of H295R was obtained at 10 mg/L and higher concentrations. Dishaw et al[12] also investigated the effect of cell vi ability only at 50 μmol/L (21.6 mg/L) TDCP using PC12 cells, and no adverse effect was found. However, Ta et al[19] reported cytotoxic results using PC12 cells that TDCP significantly decreased the cell viability from 5 μmol/L (2.2 mg/L) to 75 μmol/L (32.3 mg/L). TCEP in this study did not affect cell viability until 75 mg/L, but significantly decreased from 100
mg/L and higher concentrations, and Liu et al[18] also reported similar results from this research that less than 80% cell viability of H295R cells was achieved at 100 mg/L and greater. Ta et al[19] indicated that TCEP slightly decreased viability of PC12 cells at 15 μmol/L (4.3 mg/L) and 20
μmol/L (5.7 mg/L), whereas significantly inhibited from 40 μmol/L (11.4 mg/L) and higher concentrations. Ren et al[13] also resulted that TCEP did not affect cell viability at 0.01 mg/L, but significantly decreased from 10
mg/L using sensitive primary cultured cells. Even detection method of cytotoxicity was different from this research, Föllmann and Wober[11] als o indicated that moderate cytotoxicity was found above 10 μmol/L (2.9 mg/L) in V79 cells with the presence of S9-mix. TCPP in this research significantly decreased cell viability from 50 mg/L and upper concentrations, whereas moderate cytotoxicity was reported above 1 mmol/L (327.6 mg/L) with the presence of S9-mix[11]. Liu et al [18] could not find cytotoxic effects of TCPP even at 100 mg/L.
Fig.3 Effect of TCEP-treated HEK293 cells on cell cycle regulatory protein expression
Fig.4 Effect of TCPP-treated HEK293 cells on cell cycle regulatory protein expression
Depend on the results of EC50 conducted from this research, TCPP had four times lower EC50 value than TCEP, 58.4 mg/L and 276.8 mg/L, respectively. After using primary cultured rabbit renal proximal tubule cells, the EC50 value of TCEP was 2.9 mg/L[13]. No EC50 value of TDCP was inducted in this study due to similar percentage of cell viability from 50
mg/L to 1 000 mg/L, showing 82.3%～87.4% of the control, however, Ta et al[19] could have an EC50 value of TDCP from their results of PC12 cell viability even not mentioned in their report.
The results of cell cycle regulatory protein expression as a typical tool for studying molecular toxicity of cell growth in this research indicated that decrease of cell viability treated by TDCP might be inducted through decrease of CDK4 expression because the increase of CDK2 and cyclin E
expressions might activate DNA synthesis[20—22]. The results of cell cycle regulatory protein expressions treated by TCEP and TCPP in this study also indicated that the decrease of CDK4 expression might inhibit cell viability, showing significant decrease of the expression at 1 mg/L and 50 mg/L, whereas CDK2 and cyclin E expression showed self-discrepancy results, and further more experiments will be required. In the case of Ren et al[13], TCEP decreased cell growth via inhibiting the expression of CDK4, CDK2 and cyclin E in the primary cultured rabbit renal proximal tubule cells. Practically, it was found that decrease of cell viability might induce the decrease of cell number treated by TDCP, TCEP and TCPP, and might cause morphological changes on TCPP treated cells.
4 Conclusion
(1) TDCP, TCEP and TCPP significantly inhibited cell viability at high concentrations, but slight decrease of cell viability was only found at environmental concentrations on TDCP treated cells.
(2) The EC50 value of TCPP was four times lower than TCEP, 58.4 mg/L and 276.8 mg/L, respectively, whereas no EC50 value of TDCP was found on HEK293 cells.
(3) Morphological change was only found at 50 mg/L and 100 mg/L on TCPP treated cells, and numerical decrease was all found on TDCP, TCEP and TCPP treated cells.
(4) The three tested chemicals induced cytotoxicity might through inhibition of CDK4 regulatory protein expression.
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有机磷酸酯阻燃剂污染现状与研究进展 有机磷酸酯阻燃剂污染现状与研究进展 一、引言 随着现代科技的迅猛发展和工业生产的不断增加，阻燃剂的需求量也在持续增加。有机磷酸酯阻燃剂作为一类高效、常用的阻燃剂，应用范围广泛，但同时也带来了环境污染的问题。本文旨在探究有机磷酸酯阻燃剂的污染现状与研究进展，以期为相关领域的研究和治理提供参考。 二、有机磷酸酯阻燃剂的应用与污染源 有机磷酸酯阻燃剂具有良好的阻燃性能，广泛应用于建筑材料、电子电器、家具、汽车等领域，为提高物品的阻燃性能起到了重要作用。然而，有机磷酸酯阻燃剂的广泛应用也导致了环境中的污染。 有机磷酸酯阻燃剂的污染主要源自两个方面：一是其生产与使用过程中的排放，二是产品在使用和废弃后的释放与迁移。 1. 生产与使用过程中的排放 有机磷酸酯阻燃剂的生产过程中可能会产生一些有毒、难降解的副产物，如六溴环十二烷（HBCD）和氯代酚等。这些副产物在生产过程中会通过废水和废气排放至环境中，造成水土污染和大气污染。 除了生产过程中的排放，有机磷酸酯阻燃剂在使用过程中也存在挥发和渗透的问题。例如，在电子电器领域，电路板中使用的阻燃剂可能会逐渐释放出有机磷酸酯阻燃剂到环境中，导致环境中的污染。 2. 产品使用和废弃后的释放与迁移 有机磷酸酯阻燃剂在产品使用过程中，由于温度变化、摩擦磨
损等原因，会逐渐释放出来，并在环境中迁移。例如，室内装修中使用的含有有机磷酸酯阻燃剂的涂料、地板等，会在使用过程中逐渐释放出来，进而污染室内空气和土壤。 产品废弃后的有机磷酸酯阻燃剂也可能对环境造成污染。许多含有有机磷酸酯阻燃剂的废弃物通常被认为是危险废物，如果不经过安全处理，就可能对环境造成严重污染。 三、有机磷酸酯阻燃剂的环境效应与风险 有机磷酸酯阻燃剂在环境中的存在和迁移可能对生态环境和人类健康产生潜在的风险。 1. 生态风险 有机磷酸酯阻燃剂可能对水体生态系统产生困扰。一些研究发现，有机磷酸酯阻燃剂会对水生生物产生毒性影响，如抑制生物生长、导致畸形发育等。 此外，有机磷酸酯阻燃剂在土壤中的存在也可能对土壤生态系统造成潜在的风险。一些研究发现，有机磷酸酯阻燃剂可能导致土壤微生物的死亡和种群结构的改变，进而影响土壤中其他生物的生存和生态功能。 2. 人体健康风险 有机磷酸酯阻燃剂被普遍认为是可能对人体健康产生潜在风险的物质。一些研究发现，有机磷酸酯阻燃剂在人体内具有一定的蓄积性，并可能对神经系统、内分泌系统和免疫系统产生不利影响。 四、有机磷酸酯阻燃剂的治理与减排策略 为了减少有机磷酸酯阻燃剂的污染，需要采取一系列治理与减排策略。 1. 生产过程中的污染控制 在有机磷酸酯阻燃剂的生产过程中，需要加强废气和废水的处
气相色谱-质谱法测定塑料中7种有机磷酸酯阻燃剂的含量周小丽;郭珩;李芹;李劲光 【摘要】样品0.100 0 g置于微波萃取管中,加入乙腈15 mL,涡流振荡2 min,微波萃取20 min,冷却至室温后,收集上清液.再加入乙腈10 mL涡流振荡2 min,合并上清液.所得萃取液蒸发至近干,再用氮气吹干,用10 mL丙酮稀释.以DB-5MS色谱柱为固定相,采用气相色谱-质谱法测定上述溶液中三(2-氯乙基)磷酸酯、三(1,3-二氯丙基)磷酸酯、三(2-氯异丙基)磷酸酯、磷酸三苯酯、磷酸三对甲苯酯、磷酸三间甲苯酯和磷酸三邻甲苯酯等7种有机磷酸酯阻燃剂的含量.质谱分析中采用选择离子监测(SIM)模式.7种有机磷酸酯阻燃剂的线性范围均为1.00～100 mg·L-1,测定下限(10S/N)为0.28～1.00 mg·L-1.按标准加入法进行回收试验,回收率在85.9％～95.2％之间,相对标准偏差(n=9)均小于8.0％.%The sample (0.100 0 g) was placed into the microwave extraction tube,and 15 mL of acetonitrile was added.The mixture was oscillated whirling for 2 min and extracted by microwave for 20 min.Cooling to room temperature,the supernatant was collected.10 mL of acetonitrile was added and the mixture was oscillated whirling for 2 min.The above supernatants were combined and concentrated to dryness nearby,and then dried by N2-blowing.The residue was diluted with 10 mL of acetone.Tris(2-chloroethyl) phosphate,tris(1,3-dicloropropyl) phosphate,tris(2-chloroisopropyl) phosphate,triphenyl phosphate,tri-p-cresyl phosphate,tri-m-cresyl phosphate and tri-o-cresyl phcsphate in the above solution were determined by gas chromatography mass spectrometry (GC-MS) using DB-5MS column as the stationary phase.SIM mode was adopted in MS analysis.Linearity ranges of the 7
GCMS和LCMSMS检测木制品中三种有机磷酸酯阻燃剂 Retardants in Wood Based Panels by GCMS and LCMS/MSDong Lijun （1）Xu Meiling（1）Zhang Li（1）Li Hui（2） （1.Linyi Entry Exit Inspection and Quarantine BureauLinyi276034；2.Hubei Academy of ForestryWuhan430075 ）Abstract： In order to establish effectively organophosphorous flame retardants testing methods，we compared two methods of detecting three kinds of organophosphorous flame retardants in wood based panels combined with microwave assisted extraction in the paper. GCMS detection of TCEP，TCPP，TDCP had a good recovery distributed in the range of 870%～111.20%， RSD distributed in the range of 36%～163%， and detection limit was 29，20，14 μg?/kg1 separately； LCMS/MS detection of TCEP，TCPP，TDCP also had a good recovery distributed in the range of 827%～983%， RSD distributed in the range of 18%～108% and detection limit was 8，2，15 μg?kg1 separately. The results indicates that two methods are effective in extracting three kinds of organophosphorous flame retardants in wood based panels， and have satisfied accuracy and precision. 有机磷酸酯类阻燃剂以其出色的阻燃作用，被广泛应用于纺织、电子、家装建材等行业。随着2004年欧洲先后禁用五溴联苯醚、八溴联苯醚等溴代阻燃剂，有机磷酸酯类阻燃剂在全世界的产量和用量迅速提高，而我国2007年的产量已经超过了7万t。由于有机磷酸酯类阻燃剂以物理方式
环境中有机磷酸酯阻燃剂(TDCP、TECP和TCPP)对人胚肾 细胞(HEK 293)的影响 任相浩;陈雅岚;寇莹莹 【摘要】水环境中的微污染物有机磷酸酯如三(2-氯乙基)磷酸酯(TCEP)、三(2-氯-异丙基)磷酸酯( TCPP)和三(1,3-二氯丙基)磷酸酯(TDCP)主要用作聚氨酯泡沫塑料的阻燃剂.物理、化学和生物处理很难完全消除这些污染物.研究阻燃剂在环境水平和较高浓度下对人细胞系的细胞毒性和细胞周期效应.结果表明,在浓度为0. 001 mg/L和0. 01 mg/L时,只有TDCP具有轻微的细胞毒性,而当这三种化学物质浓度100 mg/L以上时对细胞毒性有显著的诱导作用. TCEP和TCPP的EC50分别为276. 8 mg/L和58. 4 mg/L.三种药物均能抑制CDK 4的表达,TDCP能增加CDK 2和cyclin E的表达,而TCEP和TCPP在CDK 2和cyclin E的表达上表现出自差现象. TDCP和TCEP使细胞数量减少,细胞形态发生改变.可见三种化学物质对HEK 293细胞的杀伤作用可能是通过抑制CDK 4调节蛋白而产生的. 【期刊名称】《科学技术与工程》 【年(卷),期】2019(019)010 【总页数】7页(P254-260) 【关键词】有机磷阻燃剂;微量污染物;环境水平;细胞毒性;细胞周期调节蛋白;人体细胞 【作者】任相浩;陈雅岚;寇莹莹
【作者单位】北京建筑大学环境与能源工程学院城市雨水系统与水环境教育重点实验室,北京100044;北京建筑大学环境与能源工程学院城市雨水系统与水环境教育重点实验室,北京100044;北京建筑大学环境与能源工程学院城市雨水系统与水环境教育重点实验室,北京100044 【正文语种】中文 【中图分类】X171.5 Being a major component of chlorinated organophosphate flame retardants, tris-(1,3-dichloro-isopropyl)-phosphate (TDCP), tris-(2-chloroethyl)-phosphate (TCEP) and tris-(2-chloro-isopropyl)-phosphate (TCPP) are mostly used in polyurethane foams. These retardants as typical micropollutants present in surface water, wastewater treatment plant effluent, ocean and drinking water from ng/L to μg/L level normally called as environmental level [1—3]. The elimination of these three organophosphate retardants is 0～30% by conventional wastewater treatment plant and membrane bioreactor (MBR), about 50% by ozonization, and 83%～96% by biologically active slow sand filters and GAC filtration[3—7]. Even in reverse osmosis (RO) and nanofiltration (NF), maximum removal efficiency is 95% [6,8]. Therefore, the importance of low concentration of these fire retardants remained in aquatic environment has been increasing on human health[9]. In cytotoxic study of these organophosphate retardants, only few people have investigated toxic effects using in vitro test, especially on human cells,
新兴污染物-有机磷酸酯类 摘要: 随着多溴联苯醚类阻燃剂在世界范围逐渐禁用，有机磷酸酯作为一类重要的有机磷阻燃剂和塑化剂，大量应用于塑料、纺织、家具及其他材料，从而导致了其在环境中的持续释放和分布，由此所引起的环境问题逐步引起了人们的关注。本文主要概述有机磷酸酯类阻燃剂的研究现状，包括有机磷酸酯类物质的污染现状、毒性以及分析方法。 关键词：有机磷酸酯阻燃剂环境污染毒性分析方法 1.引言 阻燃剂是一类能够阻止聚合物材料引燃或者抑制火焰传播的添加剂，有机磷酸酯(Organophosphate esters，OPEs) 是一类重要的有机磷阻燃剂(Organophosphorus flame retardants，OPFRs) ，具有阻燃效果持久，与聚合物基材相容性好，耐水、耐候、耐热以及耐迁移等特点，广泛应用于建材家装材料、纺织物品、化工以及电子电气设备中。 由于OPEs主要以添加方式而非化学键合方式加入到材料中，这增加了OPEs 类物质进入周围环境的几率因此，作为一类新有机污染物，OPEs已经受到了美国以及欧洲诸国的高度关注(如图1所示)，近几年有关OPEs的研究论文数量快速增长相关论文对OPEs的环境行为、毒性效应以及污染水平等做了初步报道。 2污染现状 2.1水体与沉积物中OPEs 表2所示为各种水体样品中OPEs的污染情。由于欧盟率先开始了对澳代阻燃剂的禁用，采用OPEs作为主要替代品，因此在欧洲多国的污水处理厂(waste water treatment plants WWTPs)中均可检出OPEs。一项针对欧洲各国污水处理厂水质情况调查显示，大多数污水处理厂的出水中可检出磷酸三氯丙酯(tri (chloropropyl) phosphate, TCPP)和磷酸三(2氯)乙酯(tri (2-hloroethyl) phosphate TCEP)，其浓度维持在几百个ng/L，并且由于TCPP难降解的特性，TCPP表现出取代TCEP成为主要的含氯OPE、污染物的趋势。研究表明德国W WTPs污水处理过程中磷酸三丁氧基乙酯(tributoxyethyl phosphate，TBEP)和磷酸三异丁酯(tri-i.sn-butyl phosphate TiBP)得到了很好的去除，去除率达80%-90%磷酸三苯酯( triphenylphosphate , TPhP)和TnBP的去除率为56%-75%而三种含氯OPE、并未得到明显去除[。在出水中检测到高浓度的TCPP表明，德国现有污水处理工艺对含氯OPE、的处理能力十分有限，只能通过活性污泥吸附有限的TCPP，需要调整现有处理工艺才能满足对TCPP的处理要求。同时，如果对吸附了TCPP的活性污泥处理不当，将造成更加严重的污染问题。在
阻燃剂分类介绍 以树脂和橡胶为基体的复合材料含有大量的有机化合物，具有一定的可燃性。阻燃剂是一类能阻止聚合物材料引燃或抑制火焰传插的添加剂。最常用的和最重要的是阻燃剂是磷、澳、氯、铢和铝的化合物。阻燃剂根据使用方法可分为添加型和反应型两大类。添加型阻燃剂主要包括磷酸酯、卤代烧及氧化锤等，它们是在复合材料加工过程中掺合于复合材料里而，使用方便，适应而大但对复合材料的性能有影响。反应型阻燃剂是在聚合物制备过程中作为一种单体原料加入聚合体系，使之通过化学反应复合到聚合物分子链上，因此对复合材料的性能影响较小，且阻燃性持久。反应型阻燃剂主要包括含磷多元醇及卤代酸肝等。 用于复合材料的阻燃剂应具备以下性能：①阻燃效率髙，能赋予复合材料良好的自熄性或难燃性：②具有良好的互容性，能与复合材料很好的相容且易分散；③具有适宜的分解温度，即在复合材料的加工温度下不分解，但是在复合材料受热分解时又能急速分解以发挥阻燃的效果； ④无毒或低毒、无臭、不污染，在阻燃过程中不产生有毒气体：⑤与复合材料并用时，不降低复合材料的力学性能、电性能、耐候性及热变形温度等：⑥耐久性好，能长期保留在复合材料的制品中，发挥其阻燃作用：⑦来源广泛价格低廉。 （1）澳系阻燃剂含澳阻燃剂包括脂肪族、脂环族、芳香族及芳香-脂肪族的含浪化合物，这类阻燃剂阻燃效率高，其阻燃效果是氯阻燃剂的两倍，相对用量少，对复合材料的力学性能几乎没有影响，并能显箸降低燃气中卤化氢的含量，而且该类阻燃剂与基体树脂互容性好，即使再苛刻的条件下也无喷岀现象。 （2）氯系阻燃剂氯系阻燃剂由于英便宜，目前仍是大量使用的阻燃剂。氯含量最高的氯化石蜡是工业上重要的阻燃剂，由于热稳泄性差，仅适用于加工温度低于200a C的复合材料，氯化脂环烧和四氯邻苯二甲酸肝热稳定性较髙，常用作不饱和树脂的阻燃剂。 （3）磷系阻燃剂、有机磷化物是添加型阻燃剂该类阻燃剂燃烧时生成的偏磷酸可形成稳定的多聚体，覆盖于复合材料表而隔绝氧和可燃物，起到阻燃作用，英阻燃效果优于浪化物，要达到同样的阻燃效果，浪化物用量为磷化物的4〜7倍。该类阻燃剂主要有磷（麟）酸酯和含卤磷酸酯及卤化磷等，广泛地用于环氧树脂、酚醛树脂、聚酯、聚碳酸酯、聚氨酯、聚氯乙烯、聚乙烯、聚丙烯、ABS等。 （4）无机阻燃剂无机阻燃剂是根据瓦化学结构习惯分出的一类阻燃剂，包括氧化鱗、氢氧化铝、氢氧化镁及硼酸锌等。 阻燃剂分类 01）、三氧化二僦：高纯2%、超细、白度98以上（添加型阻燃协效剂） 02）、三（2, 3-二浪丙基）异三聚氛酸酯：TBC、总浪疑：M%、熔点范围：100〜110°C （添加型无毒阻燃剂） 03）、三聚鼠胺編尿酸盐：MCA、含量：299 %.分解温度：440〜450°C （反应型无毒阻燃剂）04）、三浪苯酚：TBP、含：g：：2 %、熔点：M 92 °C （反应型阻燃剂） 05）、三聚磷酸铝：ATP、APW、APZ、用于生产膨胀型防火涂料、重防腐涂料（添加型无毒阻燃剂） 06）、四浪双酚A： TBBA、浪含量：2 %、熔点：180 °C （添加、反应型阻燃剂） 07）、四浪苯ST： TBPA （添加型阻燃剂） 08）、五浪甲苯：PBT （FR-5）、总浪量：＞80%.熔点：275〜284°C （添加型阻燃剂）09）、五浪联苯陆PBDPO、浪含量：62-70 （添加型阻燃剂） 10）、六浪环十二烷：HBCD （CD-75P）、总浪呈:：＞%、熔点：185〜195°C （添加型阻燃剂）11）、八澳陋：【四浪双酚A双（2, 3-二浪丙基醸）】浪含量：＞67%.熔点：＞105 °C （添加
有机磷酸酯阻燃剂的环境暴露与迁移转化研究进展 有机磷酸酯（Organophosphate Ester, OPEs）阻燃剂是 一类广泛使用于塑料制品、家具、电子设备等领域的化学物质。它们通过阻止火焰蔓延，提高了材料的火灾安全性能。然而，随着其广泛的应用，有机磷酸酯阻燃剂的环境暴露和迁移转化引起了人们的关注。 有机磷酸酯阻燃剂在使用过程中有可能从材料中逸出，并在环境中存在多种迁移转化途径。首先，OPEs可以通过气相、水相以及固相转移进入大气、水体和土壤中。研究表明，OPEs 能通过挥发、附着、溶解等方式进入大气，而且气象条件、材料性质、使用状态等因素也会影响其迁移行为。其次，OPEs 可以通过雨水沉淀、水处理过程等途径进入水体。一些OPEs 在水中的含量可达到微克级甚至高达毫克级，而且可能存在累积和生物放大的趋势。此外，固相也是OPEs的重要存在形式，它们可以通过沉积、吸附等方式进入土壤和沉积物中。相比于空气和水体，土壤和沉积物对OPEs的吸附性能更强，长期积 累可能对生态环境产生潜在影响。 目前，针对有机磷酸酯阻燃剂的环境暴露与迁移转化进行了大量的研究。研究表明，环境中的OPEs浓度与地理位置、 周边环境、使用频率等因素密切相关。一些有机磷酸酯阻燃剂如三苯磷酸酯（TBPP）和三十磷酸酯替代物（TPHPs）已经成 为环境中主要存在的OPEs类型。此外，环境中OPEs的浓度还受到生物降解和迁移转化的影响。一些微生物可降解OPEs为 较低毒性代谢产物，但一些代谢产物仍可能具有环境毒性。此外，一些OPEs还能通过光解、氧化和生物转化等方式进行迁 移转化。
有机磷酸酯阻燃剂的环境暴露与迁移转化研究对于评估其潜在环境风险和制定相关管理政策具有重要意义。近年来，一些控制有机磷酸酯阻燃剂使用的法规被相继颁布，旨在减少其对环境和健康的潜在影响。此外，科学家和工程师也在寻找绿色和环境友好型的阻燃剂替代品。例如，氮磷酸酯阻燃剂、矿物阻燃剂和纳米阻燃剂等，它们具有较低的环境风险和较好的阻燃性能。 总而言之，有机磷酸酯阻燃剂的环境暴露与迁移转化研究已经取得了一定的进展。随着对其潜在环境风险认识的不断深入和对环境保护要求的提高，研究人员将继续加大对该领域的研究力度，以推动阻燃剂的绿色替代和环境友好型设计 综上所述，有机磷酸酯阻燃剂的环境暴露与迁移转化研究已经取得了一定的进展。研究结果表明，环境中的OPEs浓度 与多种因素密切相关，包括地理位置、周围环境和使用频率等。一些OPEs如TBPP和TPHPs成为环境中主要存在的类型。此外，生物降解、迁移转化和环境毒性也对OPEs的浓度产生影响。 相关研究对于评估其潜在环境风险和制定相关管理政策具有重要意义。近年来，一些法规的制定和研究人员对绿色替代品的探索，为减少有机磷酸酯阻燃剂对环境和健康的影响提供了一定的希望。未来，需要进一步加强对该领域的研究力度，以推动阻燃剂的绿色替代和环境友好型设计
气相色谱-串联质谱法测定沉积物中有机磷酸酯 刘世龙;张华;胡晓辉;仇雁翎;朱志良;赵建夫 【摘要】比较了不同提取方法、净化方法对沉积物样品中有机磷酸酯(Organophosphate esters,OPEs)的富集、净化效果,建立了气相色谱-串联质谱法(GC-MS/MS)检测沉积物中8种OPEs的分析方法.用20 mL正己烷-丙酮混合液(1∶1,V/V)、涡流振荡+超声提取两次,Florisil固相萃取柱净化、8 mL乙酸乙酯洗脱,浓缩后将溶剂置换成正己烷,采用DB-5MS毛细管柱(30 m×0.25 mm ×0.25 μm)进行分离,质谱检测器在选择反应监测模式(SRM)下进行分析,内标法定量.结果表明,此前处理方法操作简单、溶剂耗量少;在3个添加浓度水平下,OPEs(除TEP外)的回收率在80％～ 120％之间,检出限为0.31 ～ 65 ng/L,且有良好的精密度与准确度. 【期刊名称】《分析化学》 【年(卷),期】2016(044)002 【总页数】6页(P192-197) 【关键词】有机磷酸酯;气相色谱-串联质谱;沉积物 【作者】刘世龙;张华;胡晓辉;仇雁翎;朱志良;赵建夫 【作者单位】同济大学环境科学与工程学院长江水环境教育部重点实验室,上海200092;环境科学与工程学院污染控制与资源化研究国家重点实验室,上海200092;同济大学环境科学与工程学院长江水环境教育部重点实验室,上海200092;同济大学环境科学与工程学院长江水环境教育部重点实验室,上海200092;同济大学环境
科学与工程学院长江水环境教育部重点实验室,上海200092;环境科学与工程学院 污染控制与资源化研究国家重点实验室,上海200092 【正文语种】中文 有机磷酸酯（Organophosphate esters，OPEs）是由不同烃类取代基（烷烃、 氯代烷烃、芳香烃）取代磷酸分子上的氢而形成的化合物。OPEs已经广泛应用于塑料、建筑材料、纺织品、家具等产品中。由于OPEs优异的阻燃性能以及多溴联苯醚类阻燃剂在世界范围内逐渐禁用，近年来OPEs在全球的生产量逐步增加。OPEs作为一种添加型阻燃剂，主要与化学材料键合，很容易释放到周围环境中，目前已经在多个国家与地区的多种环境介质中检出［1～14］。多项研究表明，多种OPEs具有生物毒性，氯代OPEs甚至具有致癌性［7，15～17］。 目前，对沉积物样品常用的提取方法有索氏提取［18］、超声辅助萃取法［1，19，20］、微波辅助萃取法［10，21］，加压溶剂萃取法［11，14，22，23］等。 传统的索氏提取技术成熟、稳定，但是耗费有机溶剂量大、时间长。van den Eede等［24］发现超声提取法和索氏提取法对目标物具有相似的回收率。沉积物样品有机质含量高，基质复杂，提取后需经净化才能进行仪器检测，常用的净化方法有层析柱［11，23］、凝胶渗透色谱柱［10，14，22］和固相萃取（SPE）［12，20，23］等，其中SPE操作简单方便、自动化程度高、净化效果好等优势使得其应用特别广泛。样品中有机磷酸酯的检测方法主要有气相色谱法（GC-NPD）、气相色谱质谱法（GCMS）及液相色谱串联质谱法（LC-MS/MS）等。GC-NPD对于含磷化合物尽管有较高的灵敏度，但其稳定性较差；GC-MS分析时会产生过多碎片，尤其是分析脂肪族三酯时，这些OPEs经历3次麦氏重排，会 干扰低质量离子；LC-MS/MS电喷雾电离源受样品基质干扰，从而影响灵敏度。 气相色谱-串联四级杆质谱联用（GC-MS/MS）具有更好的适用性和可靠性，采用
有机磷酸酯的环境行为及毒性 第一章有机磷酸酯概述 近十多年来，有机磷酸酯（Organophosphate esters, OPs）由于其良好的阻燃性能，广泛添加在很多产品中，例如清漆、聚氨酯泡沫、室内装潢品和纺织品[1]。多数情况下OPs以物理的方法添加到材料中，因为没有化学键的作用，在材料生产、运输和使用期间，它们都可能逐渐挥发到大气中[1]，然后分配于气相/颗粒相中，并随着空气流动迁移到室外，甚至能完成长距离的传输。 随着五溴联苯醚和八溴联苯醚的逐步禁用，为了满足各种材料的耐火要求，OPs的使用频率和用量大大增加。Nagase等人[2] 在坐垫中检测到了TBP（0.4-0.7 μg/g）、TCEP（0.8-3.1 μg/g）、TCPP（0.9-3.1 μg/g）、TDCPP（4.5-10.2 μg/g）、TPhP （4.7-23.3 μg/g）和TBEP（1.6 μg/g）。由于对防火等级要求不同，聚氨酯软泡沫中OPs的添加量存在差异，例如，美国儿童玩具的泡沫塑料中，TDCP使用频率达到36%，添加量为2.4-124mg/g（均值39.22mg/g）材料，其用量已经达到甚至超过了PBDEs。毫无疑问，使用这些玩具的婴幼儿将面临更大的OPs暴露风险[3]。在普通居民住宅中，有大量的OPs释放源，如墙纸、电视机、家具装饰材料等，例如PVC墙纸中TDCP添加含量最高可达20%，使用过程中TDCP最大释放量约为2166.8 µg/m2/h[4, 5]。Carlsson等人[6]发现电脑显示器中TPhP的含量达到了15%（w/w），电脑在使用一天后房间内TPhP的含量达到了100 ng/m3。 同样的，研究人员分析了2003-2009年间在美国购买的27件家具，发现家具中OPs用量明显增加，其中主要以含氯OPs为主，例如TDCP使用频率高达58%，添加量为1-5%；TCPP添加频率为15%，用量0.4-2.2%，仅在2004年前购买的一件家具中检测出0.5% （w/w）五溴联苯醚。住宅室内灰尘中OPs含量与这些家具的使用高度相关，TDCPP和TCPP含量分别为<90-56090ng/g和<140-5490ng/g，接近甚至超出了五溴联苯醚水平（980-44550ng/g）[7]。 TTP和TBP是液压油、润滑油中的重要成分，航空液压油中TTP添加量约1-5%，TBP含量约20%，因此在飞机维护过程中OPs挥发进入车间室内大气，含量达到mg/m3[8]，由于航空用油不溶于水，泄露或者废弃的航空油主要进入周边土壤，造成土壤中OPs的异常富集[9]。在飞机和机动车运行中，OPs直接挥发进入室外大气中，成为室外环境中OPs污染的一个重要来源[10]。 截止目前，在各种环境介质如室内空气[11]、大气[12]、污水污泥[13, 14]、表层水[15-17]、沉积物[17-19]、土壤[20]，甚至在海洋中也检测出OPs污染[19]。另一方面，随着OPs在食物链中的富集和传递[21]，OPs污染已经进入人体
有机磷酸酯单细胞测序 有机磷酸酯类化合物具有广泛的应用,包括农药、阻燃剂、塑化剂等。然而,这类物质对环境和人体健康存在潜在风险。为了更好地评估有机磷酸酯的毒性效应,研究人员开始探索单细胞测序技术在这一领域的应用。 单细胞测序是一种革命性的方法,可以在单个细胞水平上分析基因表达谱。与传统的基因组学方法相比,单细胞测序能够揭示细胞群体内的异质性,发现稀有或特异性细胞类型。这对于理解有机磷酸酯如何影响不同细胞类型和生理过程具有重要意义。 在有机磷酸酯毒性研究中,单细胞测序可用于鉴定敏感细胞类型和生物标志物。通过比较暴露组和对照组的单细胞转录组数据,研究人员能够识别出对有机磷酸酯反应最显著的细胞群。这些信息有助于阐明毒性作用机制,并为风险评估提供新的见解。 此外,单细胞测序还可以揭示有机磷酸酯诱导的适应性反应和细胞命运决定。通过对不同时间点的细胞进行测序,可以追踪细胞状态的动态变化,了解细胞如何应对化学物质胁迫。这对于预测长期健康效应和识别潜在的恢复机制至关重要。
单细胞测序产生的海量数据需要先进的生物信息学工具进行分析。聚类算法可将细胞分组为不同的亚群,而差异表达分析可鉴定各 亚群的特征基因。此外,轨迹推断方法能够重建细胞状态转换的假设 路径,揭示毒性反应的时间动力学。 尽管单细胞测序在有机磷酸酯研究中展现出巨大潜力,但仍存在 一些挑战。样本制备和测序深度可能影响数据质量和解释。此外,单 细胞数据的噪声和稀疏性给下游分析带来困难。因此,开发针对性的 质控和标准化流程至关重要。 整合单细胞转录组学与其他组学数据,如表观基因组学和蛋白质 组学,将提供更全面的毒性机制见解。多组学分析可揭示基因调控网 络和信号通路的扰动,加深对有机磷酸酯生物学效应的理解。 未来,单细胞测序有望应用于更广泛的有机磷酸酯类化合物的毒 性筛查和风险评估。建立高通量测序平台和标准化操作流程将加速 这一过程。此外,开发基于单细胞数据的预测模型和毒性评估框架, 将支持化学品安全性决策。 总的来说,单细胞测序为有机磷酸酯毒理学研究开辟了新的途径。通过揭示细胞水平的异质性和动态变化,这一技术有望加深我们对毒
人肾细胞生长分化过程中miRNA表达量的变化分析目的:分析特定miRNA在人肾细胞生长分化过程中的变化,以及miRNA在 肾细胞癌发病进展中的作用。方法:建立人肾细胞癌分化细胞系HRC-DH1与人胚肾细胞细胞系HEK293总RNA库,通过小分子RNA深度测序技术,确定人肾细胞生长、分化过程中miRNA的表达谱,并分析特定miRNA在人肾细胞生长分化过程中的变化。结果:miR-16、21、29a/b、27a、374a等miRNA在肾癌细胞分化过程中表达量下调超过两倍,而miR-368则有显著上调。在人胚肾细胞对数生长期,miR-142、16、32、20a、19a、196b等显著上调;而在接触抑制生长期,miR-19b 与miR-92a显著上调,而miR-142与miR-196b则显著下调。结论:miR-16在人肾细胞的生长分化过程中起了相反的作用,miR-196b则对于维持人肾细胞的正常生长具有重要影响。本研究为明确miRNA对于人肾细胞以及肾细胞癌的生长、发育和抑制机制奠定了基础。 标签：Micro-RNA; 人肾细胞癌; 深度测序技术 Micro-RNA(miRNA)是一类由22~23个核苷酸组成的短分子RNA,能在转录和翻译后水平调节各类基因。miRNA广泛作用于细胞生长、发育、代谢、凋亡等多种生物反应进程, 其表达量的改变与多种疾病直接相关[1-2],是近十年来生命科学领域的研究重点和热点之一。利用新的生物学技术与计算机技术探索和发现新的miRNA、研究miRNA表达谱、确定miRNA作用靶标以及研究体内miRNA 表达等,是目前miRNA研究的重点,旨在揭示miRNA在生命进程与疾病中起到的重要作用。 肾细胞癌(renal cell carcinoma,RCC),起源于肾脏的肾小管或集合管的上皮细胞,具有不同的病理类型,其发病原因还不十分明确[3]。miRNA在肾的正常发育、肾细胞癌病变过程中的作用还没有明确的报道,需要深入研究。笔者利用小分子RNA深度测序技术[4],在培养的人肾细胞癌分化细胞系HRC-DH1与人胚肾细胞细胞系HEK293中建立了相应的miRNA表达谱,分析与人胚肾细胞正常生长于癌细胞分化过程中特定miRNA的表达差异,为揭示miRNA在相应过程中的作用机制奠定基础。 1 材料与方法 1.1 实验材料 1.1.1 人肾细胞癌分化细胞系HRC-DH1与人胚肾细胞细胞系HEK293购自中科院上海细胞所。 1.1.2 RNA提取试剂Trizol购自Invitrogen公司,DMEM培养基购自GIBCO 公司,其他相关生物材料与试剂由国内生物公司提供。 1.2 实验方法 1.2.1 细胞的培养常规条件培养HRC-DH1与人胚肾细胞细胞系HEK293,37 ℃,5%CO2,DMEM培养基培养。 1.2.2 RNA的提取提取细胞总RNA前,弃培养基上清,加入PBS洗两次,弃去PBS后,加入Trizol,刮下细胞,按说明书步骤提取细胞总RNA,整个RNA提取过程,在低温或者冰上进行,以减少RNA降解。 1.2.3 小分子RNA的深度测序提取实验组细胞总RNA跑胶回收30 bp以下小分子RNA,在5’和3’分别加上一对Solexa Adaptor,这些小分子RNA再用Adaptor引物经17个循环的扩增,回收90 bp(小分子RNA+Adaptor)左右片段,送武