2,5-己二酮致人卵巢颗粒细胞凋亡及Sonic hedgehog信号通路调控机制的研究
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摘要
正已烷(n-hexane，CAS：110-54-3)，别名己烷，属于直链饱和脂肪烃类，2,5-己二酮(2,5-hexanedione，2,5-HD)是正己烷体内代谢的主要活性产物，与正己烷的毒性有关。正己烷作为有机溶剂被广泛应用的同时，如职业接触防护不当，特别是女工职业接触机会较多，会导致器官结构与功能的损伤。国内外对正己烷和2,5-HD毒性作用进行了较为广泛地研究，但主要集中在神经毒性方面，对生殖系统的毒理损伤，尤其是对女性生殖功能的影响报道较少，其毒性作用机制还有待进一步认识。2005年，有学者首次报道了来源于小鼠卵巢颗粒细胞的Hedgehog信号通路，随后研究较多的是Sonic hedgehog(SHH)。SHH信号通路对许多组织细胞增殖、分化、凋亡的调控以及在生殖腺(睾丸、卵巢)、子宫和附属性腺(前列腺、乳腺)等生殖系统中的研究是近几年的热点。因此，本研究采用体外培养的人卵巢颗粒细胞为靶细胞，通过建立和优化体外分离和培养人卵巢颗粒细胞的培养体系，研究2,5-HD致人卵巢颗粒细胞凋亡的作用机制及SHH信号通路在该过程中的调控作用，可以为明确2,5-HD对女性生殖功能的毒性损伤、作用机理以及涉及该通路的调控机制提供重要的参考依据，以期为进一步开展缓解或阻断2,5-HD致卵巢毒性损伤的研究奠定理论基础。
目的：
通过建立高效、稳定的体外分离、培养、鉴定人卵巢颗粒细胞的研究体系，从人卵巢颗粒细胞凋亡的形态学变化及与细胞凋亡有关的BCL-2家族(BCL-2、BAX)和CASPASE家族(CASPASE-3)的表达变化，探讨2,5-HD致卵巢毒性的作用机制。同时，研究Sonic hedgehog信号通路在2,5-HD致人卵巢颗粒细胞凋亡过程中的作用方式及其作用机制，为进一步研究如何缓解或阻断2,5-HD致卵巢毒性损伤提供新思路。
方法：
1.分离行辅助生殖助孕技术治疗的、符合研究纳入标准的、不孕患者的卵巢颗粒细胞，采用梯度离心、红细胞裂解液裂解及胰蛋白酶消化等方法，分离人卵巢颗粒细胞。同时，采用苏木素-伊红(HE)染色、促卵泡生成素受体(FSHR)免疫组化染色对分离细胞进行鉴定，采用CCK-8法检测细胞增殖能力，并对体外培养细胞的雌二醇(E2)、孕酮(P)分泌功能进行测定。
2.以体外培养48h的人卵巢颗粒细胞为靶细胞，采用2,5-HD终浓度为0、16μmol/L、64μmol/L和256μmol/L的DMEM培养液体外染毒24h，通过采用HE染色、Hoechst33342荧光染色、CASPASE-3p17免疫组化染色、透射电镜以及流式细胞仪Annexin V-FITC/PI双染法检测2,5-HD诱导人卵巢颗粒细胞凋亡的改变。再进一步采用realtime PCR和Western blot法，分别检测与细胞凋亡有关的BCL-2家族(BCL-2、BAX)和CASPASE家族(CASPASE-3)的mRNA、蛋白水平随着2,5-HD染毒浓度的增加其表达量的变化情况。
3.选择研究致人卵巢颗粒细胞凋亡效应最显著的256μmol/L2,5-HD体外染毒24h过程的颗粒细胞和正常对照组颗粒细胞为研究对象，采用realtime PCR，测定内源性SHH信号通路在2,5-HD作用24h致人卵巢颗粒细胞凋亡过程中的mRNA的表达改变；采用Western blot法，测定内源性SHH信号通路主要效应分子SHH、GLI1在2,5-HD作用24h致人卵巢颗粒细胞凋亡过程中的蛋白表达改变情况。同时，通过测定外源性重组SHH因子和SHH信号通路阻滞剂Cyclopamine对SHH信号通路的激活或阻断，来进一步验证SHH信号通路对2,5-HD致人卵巢颗粒细胞凋亡率的影响，以及对抗凋亡基因BCL-2和凋亡基因BAX mRNA、蛋白的影响。
结果：
1.人卵巢颗粒细胞分离、原代培养与鉴定体系的建立：(1)台盼蓝染色显示实验分离的人卵巢颗粒细胞存活率>90%，细胞表达FSHR的阳性率>95%。(2)光镜下观察到，人卵巢颗粒细胞体外生长速度较慢，培养2d后细胞明显增殖，细胞与细胞之间有丝状突起相连；第6-7d时细胞逐渐变形、退化。(3)采用CCK-8法测定细胞增殖曲线显示，人卵巢颗粒细胞随着体外培养时间的延长而增殖,培养24h时细胞处于贴壁生长期，在培养第3-5d时达到分裂高峰，随后第6-7d细胞开始逐渐退化。(4)体外培养的人卵巢颗粒细胞在重组人促卵泡激素(rFSH)作用下可明显促进E2的分泌，但分泌孕酮(P)的能力与rFSH的作用无关，体外分离、原代培养的人卵巢颗粒细胞保持了原有细胞的功能特性。
2.2,5-HD致人卵巢颗粒细胞凋亡机制的研究：本研究建立了2,5-HD诱导的人卵巢颗粒细胞凋亡模型，由相差显微镜、光镜、荧光显微镜和透射电镜可观察到特征性的凋亡形态学改变，且随着2,5-HD染毒浓度的增加出现凋亡形态学改变的人卵巢颗粒细胞会逐渐增多。流式细胞仪Annexin V-FITC/PI双染法结果显示，2,5-HD终浓度为64μmol/L、256μmol/L时可显著增加颗粒细胞的凋亡率。realtime PCR结果显示，随着2,5-HD染毒浓度的增加，BCL-2mRNA水平呈下降趋势，BAX、CASPASE-3mRNA水平呈上升趋势。Western blot与realtime PCR结果相一致，表现为随着2,5-HD染毒浓度的增加，BCL-2蛋白表达呈下降趋势，凋亡蛋白BAX、凋亡下游执行蛋白CASPASE-3(p17)表达呈上升趋势。
3. Sonic hedgehog信号通路在2,5-HD致人卵巢颗粒细胞凋亡过程中的调控作用：(1)对照组人卵巢颗粒细胞在体外培养过程中，有少量SHH信号通路组分表达，但随时间改变无明显改变；在2,5-HD作用24h过程中，颗粒细胞SHH、PTCH1、SMO以及GLI1mRNA水平在3h、6h时分别与对照组比较差异显著；SHH、SMO和GLI1mRNA水平在24h时均有下降；GLI2和GLI3mRNA水平与对照组比较在不同时间差异均无统计学意义；人卵巢颗粒细胞SHH、PTCH1、SMO以及GLI1mRNA水平在3h、6h时升高分别为对照组的2-5倍、5-8倍，随后于24h时降到低值。(2)与realtime PCR结果相一致，对照组人卵巢颗粒细胞在体外培养过程中，SHH信号通路主要效应分子SHH、GLI1蛋白有少量表达，随时间改变无明显差异；在2,5-HD作用24h过程中，颗粒细胞SHH和GLI1蛋白水平在3h、6h时升高分别与对照组比较差异显著，在24h时均有下降。(3)外源性SHH因子可增加人卵巢颗粒细胞的存活率，减少2,5-HD作用导致的细胞凋亡；用外源性Cyclopamine阻滞SHH信号通路会增加人卵巢颗粒细胞的凋亡率。(4)在2,5-HD致人卵巢颗粒细胞凋亡过程中采用外源性重组SHH因子处理会导致抗凋亡基因BCL-2表达上调，凋亡基因BAX表达下降。
结论：
1.采用本研究所建立的方法可获得生长活性较好、细胞纯度高及稳定的人卵巢颗粒细胞，用HE染色和FSHR免疫组化染色法可简便快速地鉴定人卵巢颗粒细胞。
2.2,5-HD通过诱导人卵巢颗粒细胞BCL-2家族中凋亡基因(BAX)表达上升和抗凋亡基因(BCL-2)表达下降，引起凋亡下游执行蛋白CASPASE-3(p17)表达上升，进而导致颗粒细胞的凋亡增加，有可能是其致卵巢功能受损的机制之一，明确BCL-2家族对2,5-HD致人卵巢颗粒细胞凋亡的作用机制，可以为女性生殖毒性损伤的临床诊断、确定分子治疗靶点的研究提供参考依据。
3.体外培养的人卵巢颗粒细胞有少量SHH信号通路组分表达。在2,5-HD致人卵巢颗粒细胞凋亡过程中SHH信号通路会被激活，起到了降低颗粒细胞凋亡的保护性作用，其作用机制有可能是通过调节抗凋亡基因BCL-2和凋亡基因BAX的表达改变，来阻止或缓解2,5-HD诱导的细胞凋亡。
n-Hexane (CAS:110-54-3), alias hexane, are straight-chain saturated fattyhydrocarbon. As the main active metabolite of n-hexane in the human body, thetoxicity of2,5-hexanedione (2,5-HD) and n-hexane are related. n-Hexane is widelyused as an organic solvent. n-Hexane toxicity causes serious harm in workers,particularly in women. The toxicity of n-hexane and2,5-HD has been extensivelyresearched, but research has primarily concentrated on neural toxicity. Damage to thereproductive system, especially for female reproductive function, has been lessfrequently reported. In2005, some scholars had the first report of the Hedgehogsignaling pathway derived from mouse ovarian granulosa cells, in which sonichedgehog (SHH) signaling pathway was more researched. SHH signaling pathwaydrives cell proliferation, directs cell differentiation and promotes cell away fromapoptosis. The emerging roles for SHH signaling pathway in the gonads (testis, ovary),uterus and accessory sex glands such as the prostate and mammary gland are beingfocused on in recent years. Therefore, this study established the efficient and stableprimary culture in vitro and identification methods of human ovarian granulosa cells.We used human ovarian granulosa cells as target cells to study whether the2,5-HDinduced cell apoptosis and in which the mechanism of SHH signaling pathway. In theconclusion, we could clarify the toxic effects and mechanism of2,5-HD on femalereproductive function, as well as the regulation mechanism involved in which, so could provide an important reference about how to ease or blocked2,5-HD toxicitydamage to ovarian.
Objective:
To establish the efficient and stable primary culture in vitro and identificationmethods of human ovarian granulosa cells, so to lay the foundation for in vitroreproductive toxicology studies. The other purpose was to investigate the mechanismsof human ovarian toxicity induced by2,5-HD through the morphological changes andapoptosis in human ovary granulosa cells and BCL-2family (BCL-2、BAX) andCASPASE family (CASPASE-3) expression changes in which. And to investigate theSHH signaling pathway in2,5-HD induced apoptosis in human ovarian granulosacells and its mechanism, in order to provide new ideas about how to ease or blocked2,5-HD toxicity damage to ovarian.
Methods:
1. Primary granulosa cells were obtained from women who were meeting theinclusion criteria and undergoing artificial reproductive technology (ART). Humangranulosa cells were collected by gradient centrifugation, red blood cell lysis withlysis of red blood cells and trypsin digestion. The hematoxylin-eosin (HE) stainingand follicle-stimulating hormone (FSHR) immunohistochemistry methods were usedto identify ovarian granulosa cells. CCK-8method was used to assay the cellproliferation. Meanwhile, in vitro granulosa cell secretion of estradiol (E2) andprogesterone (P) were determined.
2. The study used cultured human granulosa cells as target cells, which wereexposed to0,16μmol/L,64μmol/L, and256μmol/L2,5-HD in vitro for24h. The HEstaining, Hoechst33342staining, transmission electron microscopy, and flowcytometry using FITC-Annexin V/PI double staining were used to prove that2,5-HDcould lead to apoptosis in human ovarian granulosa cells. Realtime quantitative PCRand Western blot analysis were used to detect changes in the expression of theapoptosis-related BCL-2family (BCL-2, BAX) and CASPASE family (CASPASE-3) with increasing2,5-HD concentration.
3. The study used cultured human granulosa cells as target cells, which wereexposed to0and256μmol/L2,5-HD in vitro for24h. Realtime PCR and Western blotanalysis were used to detect the endogenous expression of SHH signaling in theprocess of2,5-HD-induced granulosa cells apoptosis. At the same time, the cellapoptosis were determined when the activation or blockade of SHH signaling pathwayby the recombinant SHH and cyclopamine, as well as the impact on anti-apoptoticgene BCL-2and apoptotic gene BAX.
Results:
1. Separaton, Primary culture and identification of human ovarian granulosa cells:(1) trypan blue staining showed that the granulosa cell viability over90%by thismethod. Cell purity was above95%by FSHR immunohistochemical identification.(2)By the light microscopy, cells in vitro were slow to develop, and two days later thecells proliferated significantly. At the6-7d cells gradually deformed and degradated.(3) CCK-8curve showed cultured human ovarian granulosa cells in24h at adherentgrowing season. And cultured cells during the first3-5d were to reach the splittingpeak, then began to degenerate at6-7d.(4) Cultured human granulosa cells under theeffect of recombinant FSH (rFSH) could significantly promote the secretion of E2, butthe secretion of P ability had nothing to do with the role of rFSH. Primary culturedhuman ovarian granulosa cells were maintaining the original cell characteristics.
2. The mechanism of2,5-hexanedione inducing human ovarian granulosa cellapoptosis: this study established a human ovarian granulosa cell apoptosis modelinduced by2,5-HD.2,5-HD was demonstrated to cause significant apoptosis of humanovarian granulosa cells in a dose-dependent manner. The results showed that withincreasing2,5-HD doses, the expression of BCL-2decreased. However, a markeddose-dependent increase in the expression of BAX and active CASPASE-3(p17) wasobserved in human ovarian granulosa cells.
3. The regulation mechanism of Sonic Hedgehog signaling pathways in the processof2,5-hexanedione inducing apoptosis in human ovarian granulosa cells:(1) Control group of human ovarian granulosa cells in vitro culture, the expression of a smallamount of SHH signaling pathway components, but no significant difference inchange over time. In the2,5-HD-induced apoptosis in human ovarian granulosa cells,SHH, PTCH1, SMO, and GLI1mRNA levels in3h,6h, respectively compared withthe control group, were significantly different. SHH、SMO and GLI1mRNA levels in24h were lower. GLI2and GLI3mRNA at different times compared with the controlgroup were not statistically significant.(2) Results were consistent with the realtimePCR, SHH, GLI1protein of human ovarian granulosa cells in3h and6h wereelevated, and decreased in24h.(3) The exogenous SHH could increase the survivalrate of human ovarian granulosa cells and reduce cells apoptosis. The cyclopaminecould increase the cells apoptosis by blocking SHH signaling pathway.(4) In the2,5-HD-induced apoptosis in human ovarian granulosa cells, exogenous recombinantSHH could lead to increase expression of BCL-2and decrease expression of BAX.
Conclusions:
1. Good growth activity, cell purity and more stable human ovarian granulosa cellscould be obtained by the method used in this study. Human ovarian granulosa cellscould be simply and rapidly identified by HE staining and FSHR immuno-histochemistry methods.
2. These results suggest that the mechanisms of2,5-HD causing increased apoptosisin human ovarian granulosa cells might be a marked dose-dependent on the expressionof BCL-2decreased and the expression of BAX and active CASPASE-3(p17)increased.
3. Cultured human granulosa cells expressed a small amount of SHH signalingpathway components. SHH signaling pathway was activated and could reduce theapoptosis rate of granulosa cells during the2,5-HD-induced granulosa cells apoptosis.Its mechanism might be by regulating anti-apoptotic gene BCL-2and apoptosis geneBAX to prevent or alleviate the2,5-HD-induced granulosa cells apoptosis.
目的：
通过建立高效、稳定的体外分离、培养、鉴定人卵巢颗粒细胞的研究体系，从人卵巢颗粒细胞凋亡的形态学变化及与细胞凋亡有关的BCL-2家族(BCL-2、BAX)和CASPASE家族(CASPASE-3)的表达变化，探讨2,5-HD致卵巢毒性的作用机制。同时，研究Sonic hedgehog信号通路在2,5-HD致人卵巢颗粒细胞凋亡过程中的作用方式及其作用机制，为进一步研究如何缓解或阻断2,5-HD致卵巢毒性损伤提供新思路。
方法：
1.分离行辅助生殖助孕技术治疗的、符合研究纳入标准的、不孕患者的卵巢颗粒细胞，采用梯度离心、红细胞裂解液裂解及胰蛋白酶消化等方法，分离人卵巢颗粒细胞。同时，采用苏木素-伊红(HE)染色、促卵泡生成素受体(FSHR)免疫组化染色对分离细胞进行鉴定，采用CCK-8法检测细胞增殖能力，并对体外培养细胞的雌二醇(E2)、孕酮(P)分泌功能进行测定。
2.以体外培养48h的人卵巢颗粒细胞为靶细胞，采用2,5-HD终浓度为0、16μmol/L、64μmol/L和256μmol/L的DMEM培养液体外染毒24h，通过采用HE染色、Hoechst33342荧光染色、CASPASE-3p17免疫组化染色、透射电镜以及流式细胞仪Annexin V-FITC/PI双染法检测2,5-HD诱导人卵巢颗粒细胞凋亡的改变。再进一步采用realtime PCR和Western blot法，分别检测与细胞凋亡有关的BCL-2家族(BCL-2、BAX)和CASPASE家族(CASPASE-3)的mRNA、蛋白水平随着2,5-HD染毒浓度的增加其表达量的变化情况。
3.选择研究致人卵巢颗粒细胞凋亡效应最显著的256μmol/L2,5-HD体外染毒24h过程的颗粒细胞和正常对照组颗粒细胞为研究对象，采用realtime PCR，测定内源性SHH信号通路在2,5-HD作用24h致人卵巢颗粒细胞凋亡过程中的mRNA的表达改变；采用Western blot法，测定内源性SHH信号通路主要效应分子SHH、GLI1在2,5-HD作用24h致人卵巢颗粒细胞凋亡过程中的蛋白表达改变情况。同时，通过测定外源性重组SHH因子和SHH信号通路阻滞剂Cyclopamine对SHH信号通路的激活或阻断，来进一步验证SHH信号通路对2,5-HD致人卵巢颗粒细胞凋亡率的影响，以及对抗凋亡基因BCL-2和凋亡基因BAX mRNA、蛋白的影响。
结果：
1.人卵巢颗粒细胞分离、原代培养与鉴定体系的建立：(1)台盼蓝染色显示实验分离的人卵巢颗粒细胞存活率>90%，细胞表达FSHR的阳性率>95%。(2)光镜下观察到，人卵巢颗粒细胞体外生长速度较慢，培养2d后细胞明显增殖，细胞与细胞之间有丝状突起相连；第6-7d时细胞逐渐变形、退化。(3)采用CCK-8法测定细胞增殖曲线显示，人卵巢颗粒细胞随着体外培养时间的延长而增殖,培养24h时细胞处于贴壁生长期，在培养第3-5d时达到分裂高峰，随后第6-7d细胞开始逐渐退化。(4)体外培养的人卵巢颗粒细胞在重组人促卵泡激素(rFSH)作用下可明显促进E2的分泌，但分泌孕酮(P)的能力与rFSH的作用无关，体外分离、原代培养的人卵巢颗粒细胞保持了原有细胞的功能特性。
2.2,5-HD致人卵巢颗粒细胞凋亡机制的研究：本研究建立了2,5-HD诱导的人卵巢颗粒细胞凋亡模型，由相差显微镜、光镜、荧光显微镜和透射电镜可观察到特征性的凋亡形态学改变，且随着2,5-HD染毒浓度的增加出现凋亡形态学改变的人卵巢颗粒细胞会逐渐增多。流式细胞仪Annexin V-FITC/PI双染法结果显示，2,5-HD终浓度为64μmol/L、256μmol/L时可显著增加颗粒细胞的凋亡率。realtime PCR结果显示，随着2,5-HD染毒浓度的增加，BCL-2mRNA水平呈下降趋势，BAX、CASPASE-3mRNA水平呈上升趋势。Western blot与realtime PCR结果相一致，表现为随着2,5-HD染毒浓度的增加，BCL-2蛋白表达呈下降趋势，凋亡蛋白BAX、凋亡下游执行蛋白CASPASE-3(p17)表达呈上升趋势。
3. Sonic hedgehog信号通路在2,5-HD致人卵巢颗粒细胞凋亡过程中的调控作用：(1)对照组人卵巢颗粒细胞在体外培养过程中，有少量SHH信号通路组分表达，但随时间改变无明显改变；在2,5-HD作用24h过程中，颗粒细胞SHH、PTCH1、SMO以及GLI1mRNA水平在3h、6h时分别与对照组比较差异显著；SHH、SMO和GLI1mRNA水平在24h时均有下降；GLI2和GLI3mRNA水平与对照组比较在不同时间差异均无统计学意义；人卵巢颗粒细胞SHH、PTCH1、SMO以及GLI1mRNA水平在3h、6h时升高分别为对照组的2-5倍、5-8倍，随后于24h时降到低值。(2)与realtime PCR结果相一致，对照组人卵巢颗粒细胞在体外培养过程中，SHH信号通路主要效应分子SHH、GLI1蛋白有少量表达，随时间改变无明显差异；在2,5-HD作用24h过程中，颗粒细胞SHH和GLI1蛋白水平在3h、6h时升高分别与对照组比较差异显著，在24h时均有下降。(3)外源性SHH因子可增加人卵巢颗粒细胞的存活率，减少2,5-HD作用导致的细胞凋亡；用外源性Cyclopamine阻滞SHH信号通路会增加人卵巢颗粒细胞的凋亡率。(4)在2,5-HD致人卵巢颗粒细胞凋亡过程中采用外源性重组SHH因子处理会导致抗凋亡基因BCL-2表达上调，凋亡基因BAX表达下降。
结论：
1.采用本研究所建立的方法可获得生长活性较好、细胞纯度高及稳定的人卵巢颗粒细胞，用HE染色和FSHR免疫组化染色法可简便快速地鉴定人卵巢颗粒细胞。
2.2,5-HD通过诱导人卵巢颗粒细胞BCL-2家族中凋亡基因(BAX)表达上升和抗凋亡基因(BCL-2)表达下降，引起凋亡下游执行蛋白CASPASE-3(p17)表达上升，进而导致颗粒细胞的凋亡增加，有可能是其致卵巢功能受损的机制之一，明确BCL-2家族对2,5-HD致人卵巢颗粒细胞凋亡的作用机制，可以为女性生殖毒性损伤的临床诊断、确定分子治疗靶点的研究提供参考依据。
3.体外培养的人卵巢颗粒细胞有少量SHH信号通路组分表达。在2,5-HD致人卵巢颗粒细胞凋亡过程中SHH信号通路会被激活，起到了降低颗粒细胞凋亡的保护性作用，其作用机制有可能是通过调节抗凋亡基因BCL-2和凋亡基因BAX的表达改变，来阻止或缓解2,5-HD诱导的细胞凋亡。
n-Hexane (CAS:110-54-3), alias hexane, are straight-chain saturated fattyhydrocarbon. As the main active metabolite of n-hexane in the human body, thetoxicity of2,5-hexanedione (2,5-HD) and n-hexane are related. n-Hexane is widelyused as an organic solvent. n-Hexane toxicity causes serious harm in workers,particularly in women. The toxicity of n-hexane and2,5-HD has been extensivelyresearched, but research has primarily concentrated on neural toxicity. Damage to thereproductive system, especially for female reproductive function, has been lessfrequently reported. In2005, some scholars had the first report of the Hedgehogsignaling pathway derived from mouse ovarian granulosa cells, in which sonichedgehog (SHH) signaling pathway was more researched. SHH signaling pathwaydrives cell proliferation, directs cell differentiation and promotes cell away fromapoptosis. The emerging roles for SHH signaling pathway in the gonads (testis, ovary),uterus and accessory sex glands such as the prostate and mammary gland are beingfocused on in recent years. Therefore, this study established the efficient and stableprimary culture in vitro and identification methods of human ovarian granulosa cells.We used human ovarian granulosa cells as target cells to study whether the2,5-HDinduced cell apoptosis and in which the mechanism of SHH signaling pathway. In theconclusion, we could clarify the toxic effects and mechanism of2,5-HD on femalereproductive function, as well as the regulation mechanism involved in which, so could provide an important reference about how to ease or blocked2,5-HD toxicitydamage to ovarian.
Objective:
To establish the efficient and stable primary culture in vitro and identificationmethods of human ovarian granulosa cells, so to lay the foundation for in vitroreproductive toxicology studies. The other purpose was to investigate the mechanismsof human ovarian toxicity induced by2,5-HD through the morphological changes andapoptosis in human ovary granulosa cells and BCL-2family (BCL-2、BAX) andCASPASE family (CASPASE-3) expression changes in which. And to investigate theSHH signaling pathway in2,5-HD induced apoptosis in human ovarian granulosacells and its mechanism, in order to provide new ideas about how to ease or blocked2,5-HD toxicity damage to ovarian.
Methods:
1. Primary granulosa cells were obtained from women who were meeting theinclusion criteria and undergoing artificial reproductive technology (ART). Humangranulosa cells were collected by gradient centrifugation, red blood cell lysis withlysis of red blood cells and trypsin digestion. The hematoxylin-eosin (HE) stainingand follicle-stimulating hormone (FSHR) immunohistochemistry methods were usedto identify ovarian granulosa cells. CCK-8method was used to assay the cellproliferation. Meanwhile, in vitro granulosa cell secretion of estradiol (E2) andprogesterone (P) were determined.
2. The study used cultured human granulosa cells as target cells, which wereexposed to0,16μmol/L,64μmol/L, and256μmol/L2,5-HD in vitro for24h. The HEstaining, Hoechst33342staining, transmission electron microscopy, and flowcytometry using FITC-Annexin V/PI double staining were used to prove that2,5-HDcould lead to apoptosis in human ovarian granulosa cells. Realtime quantitative PCRand Western blot analysis were used to detect changes in the expression of theapoptosis-related BCL-2family (BCL-2, BAX) and CASPASE family (CASPASE-3) with increasing2,5-HD concentration.
3. The study used cultured human granulosa cells as target cells, which wereexposed to0and256μmol/L2,5-HD in vitro for24h. Realtime PCR and Western blotanalysis were used to detect the endogenous expression of SHH signaling in theprocess of2,5-HD-induced granulosa cells apoptosis. At the same time, the cellapoptosis were determined when the activation or blockade of SHH signaling pathwayby the recombinant SHH and cyclopamine, as well as the impact on anti-apoptoticgene BCL-2and apoptotic gene BAX.
Results:
1. Separaton, Primary culture and identification of human ovarian granulosa cells:(1) trypan blue staining showed that the granulosa cell viability over90%by thismethod. Cell purity was above95%by FSHR immunohistochemical identification.(2)By the light microscopy, cells in vitro were slow to develop, and two days later thecells proliferated significantly. At the6-7d cells gradually deformed and degradated.(3) CCK-8curve showed cultured human ovarian granulosa cells in24h at adherentgrowing season. And cultured cells during the first3-5d were to reach the splittingpeak, then began to degenerate at6-7d.(4) Cultured human granulosa cells under theeffect of recombinant FSH (rFSH) could significantly promote the secretion of E2, butthe secretion of P ability had nothing to do with the role of rFSH. Primary culturedhuman ovarian granulosa cells were maintaining the original cell characteristics.
2. The mechanism of2,5-hexanedione inducing human ovarian granulosa cellapoptosis: this study established a human ovarian granulosa cell apoptosis modelinduced by2,5-HD.2,5-HD was demonstrated to cause significant apoptosis of humanovarian granulosa cells in a dose-dependent manner. The results showed that withincreasing2,5-HD doses, the expression of BCL-2decreased. However, a markeddose-dependent increase in the expression of BAX and active CASPASE-3(p17) wasobserved in human ovarian granulosa cells.
3. The regulation mechanism of Sonic Hedgehog signaling pathways in the processof2,5-hexanedione inducing apoptosis in human ovarian granulosa cells:(1) Control group of human ovarian granulosa cells in vitro culture, the expression of a smallamount of SHH signaling pathway components, but no significant difference inchange over time. In the2,5-HD-induced apoptosis in human ovarian granulosa cells,SHH, PTCH1, SMO, and GLI1mRNA levels in3h,6h, respectively compared withthe control group, were significantly different. SHH、SMO and GLI1mRNA levels in24h were lower. GLI2and GLI3mRNA at different times compared with the controlgroup were not statistically significant.(2) Results were consistent with the realtimePCR, SHH, GLI1protein of human ovarian granulosa cells in3h and6h wereelevated, and decreased in24h.(3) The exogenous SHH could increase the survivalrate of human ovarian granulosa cells and reduce cells apoptosis. The cyclopaminecould increase the cells apoptosis by blocking SHH signaling pathway.(4) In the2,5-HD-induced apoptosis in human ovarian granulosa cells, exogenous recombinantSHH could lead to increase expression of BCL-2and decrease expression of BAX.
Conclusions:
1. Good growth activity, cell purity and more stable human ovarian granulosa cellscould be obtained by the method used in this study. Human ovarian granulosa cellscould be simply and rapidly identified by HE staining and FSHR immuno-histochemistry methods.
2. These results suggest that the mechanisms of2,5-HD causing increased apoptosisin human ovarian granulosa cells might be a marked dose-dependent on the expressionof BCL-2decreased and the expression of BAX and active CASPASE-3(p17)increased.
3. Cultured human granulosa cells expressed a small amount of SHH signalingpathway components. SHH signaling pathway was activated and could reduce theapoptosis rate of granulosa cells during the2,5-HD-induced granulosa cells apoptosis.Its mechanism might be by regulating anti-apoptotic gene BCL-2and apoptosis geneBAX to prevent or alleviate the2,5-HD-induced granulosa cells apoptosis.
引文
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