胸腺活化调节趋化因子及其受体在早孕绒毛组织中的表达特征及生物学功能

Expression and function of chemokine TARC/CCR4 at fetal-maternal interface in first trimester

【摘要】目的探讨早孕绒毛组织中胸腺活化调节趋化因子（TARC）及其特异性受体4（CCR4）的表达特征及生物学功能。方法收集正常早孕（孕6—8周）妇女人工流产术后的绒毛组织，采用逆转录（RT）PCR技术检测TARC、CCR4的mRNA表达水平；免疫组化法检测TARC、CCR4的蛋白表达水平。原代分离、培养绒毛外细胞滋养细胞，免疫荧光法检测TARC、CCR4蛋白在细胞水平的表达；体外侵袭实验检测不同浓度（10、25、50、100ng／m1）的重组蛋白TARC（rhTARC）与无血清RPMI1640培养基（对照组）对绒毛外细胞滋养细胞系HTR8／SVneo细胞侵袭能力的影响；阻断试验检测TARC对细胞侵袭能力影响的特异性，实验分4组：对照组、100ng／ml TARC组、100ng／mlrhTARC＋20μg/mlTARC抗体组、100ng／ml TARC＋20μg／mlIgG组，检测各组HTR8／SVneo细胞的侵袭能力变化；蛋白印迹法检测100ng／mlrhTARC组整合素击及整合素B1蛋白表达水平。结果（1）体内试验：人早孕绒毛组织中均有TARC及CCR4mRNA的表达；TARC蛋白主要表达于细胞滋养细胞、合体滋养细胞及滋养层柱的远端，CCR4蛋白特异性地表达于绒毛外间质细胞滋养细胞。（2）体外试验：①免疫荧光法检测显示，TARC、CCR4蛋白在绒毛外细胞滋养细胞中的表达均呈阳性；②体外侵袭实验显示，HTR8／SVneo细胞经10、25、50、100ng／ml浓度的ATARC处理后，细胞的侵袭能力逐渐增加，穿膜细胞数分别为（142±31）、（161±46）、（201±30）、（312±-48）个，对照组为（117±33）个，与25ng／ml TARC处理时的穿膜细胞数比较，差异有统计学意义（P〈0．05）；100ng／ml的 rhTARC处理时，侵袭能力达高峰（P〈0．01）；③阻断试验结果显示，100ng／mlrhTARC组、100ng／ml TARC＋20μg/ml TARC抗体组、100ng/ml rhTARC＋20μg/ml IgG组和对照组侵袭到小室对侧的细胞数分别为（313±47）、（113±41）、（287±75）、（128±23）个；④免疫印迹法检测结果显示，与对照组比较，100rig／mlrhTARC组整合素硝的蛋白表达水平明显升高，整合素β1的蛋白表达水平也有所升高，差异均有统计学意义（P〈0．01，P〈0．05）。结论TARC特异性地表达于人早孕绒毛，并通过上调整合素α5及整合素β1的蛋白表达增加滋养细胞的侵袭能力，可能在滋养细胞分化及胎盘形成过程中发挥重要作用。

Objective To investigate the expression and function of thymus and activation regulated chemokine （TARC） and its special receptor CCR4 at placenta villous in the first trimester placenta villous. Methods Placenta villous was collected from healthy women undergoing artificial abortion at 6 to 8 weeks of gestation, mRNA levels of TARC, CCR4 were analyzed using semi-quantitative reverse transcription （RT）-PCR methods. Immnnohistochemistry assay was used to assess the protein localization and expression of TARC, CCR4. Additionally, extravillous cytotrophoblasts were isolated and cultured. Expression of TARC and CCR4 was measured by immunofluorescence assay. Invasion of cell line HTRS/SVneo was analyzed by transwell assay at concentration of 10,25,50 and 100 ng/ml of TARC matched with RPMI 1640 fetal bovine serum free culture medium as control group. In the mean time, blocking experiment was also added to detect TARC regulating cell invasion, which were classified into four groups：control, 100 ng/ml rhTARC ,20 μg/ml anti-TARC ± 100 ng/ml rhTARC, 100 ng/ml rhTARC ± 20 μg/ml IgG. The influence of 100 ng/ml TARC on expression level of integrin-cL5 and integrin-IM were measured by using western-blot assay. Results （ 1 ） In vivo assay：expression of TARC and CCR4 mRNA were detectable in first trimester placenta villous, TARC protein was localized in cytotrophoblasts, syncytiotrophoblasts and cell column especially on the distal portion, while CCR4 protein was localized on invading interstitial cytotrophobalsts. （2） In vitro assay： （~） TARC ,CCR4 was also expressed in primary isolated extravillous cytotrophoblasts by immunofluorescence assay; （）Matrigel invasion assay demonstrated that TARC had specific dose dependent sfimulatory effects on the cells invading through the matrigel precoated filter, the number of cells migration into the lower chamber were：142 ±31 at 10 ng/ml group, 161 ±46 at 25 ng/ml group, 201 ±30 at 50 ng/ml group, 312 ±48 at 100 ng/ml group, 117 ± 33 at control group, the significant response observed from 25 ng/ml （ P 〈 0.05 ） and reached a peak effect at 100 ng/ml （ P 〈 0. 01 ） ; （2） Blocking experiment demonstrated that when trophoblast invasion was monitored in response to TARC neutralizing antibody （15 μg/ml） together with rhTARC 100 ng/ml. The stimulatory activity of rhTARC was completely overcome, with the ceils invasion into the lower chambers were 100 ng/ml rhTARC,20 μg/ml anti-TARC ± 100 ng/ml rhTARC, 100 ng/ml rhTARC ±20 μg/ml IgG, control：313 ±47, 113 ±41, 287 ±75 and 128 ±23, respectively;@Western-blot assay demonstrated that if cells were treated with 100 ng/ml rhTARC, the expression of integrin-ct5 were significantly increased（ P 〈 0. 01 ）, integrin-131 level also increased when compared with control（ P 〈 0. 05 ）. Conclusion TARC was expressed specifically at human fetal-maternal interface. Trophoblast invasion and migration mainly was regulated by up-regulation integrin-α5 and integrin-β1, which plays an role in trophoblasts differentiation and placentation.