【作者】 胡翔；

【导师】 陆华；

【作者基本信息】 成都中医药大学 ， 中医妇科学， 2013， 博士

【摘要】 目的：观察含补肾中药鼠血清对人早孕流产胚胎脐带血管周围干细胞(FTM-PVCs)体外分化为类卵细胞的影响第一部分：人FTM-PVCs体外分化为类卵细胞潜能的研究和体外干细胞研究平台的建立方法：1、不同方法诱导人FTM-PVCs体外分化为类卵细胞的研究：人FTM-PVCs的分离纯化后,将细胞诱导为拟胚体,以各种比例人卵泡液诱导拟胚体分化为类卵细胞：在诱导分化不同时间段,比较各组细胞形态学变化,免疫组化法检测细胞卵细胞和透明带标志物的表达,酶联免疫法检测不同时间段培养液中的E2含量。以各种诱导方案诱导贴壁培养的人FTM-PVCs分化为类卵细胞：分为25%人卵泡液+FSH、LH培养组；25%人卵泡液+FSH、LH+E2培养组；25%人卵泡液+FSH、LH+E2+叶酸培养组；25%人卵泡液+FSH、 LH+E2+IVM培养液(HTF培养基+10%血清白蛋白代用品)培养组,FSH、 LH+E2培养组；人颗粒细胞共培养组,显微镜下观察各组细胞形态学变化并拍照。2、体外干细胞研究平台的建立：细胞免疫组化法、细胞免疫荧光法检测25%人卵泡液+FSH、LH+E2诱导人FTM-PVCs分化后各种特异性标志物的表达,RT-PCR半定量检测诱导分化后的人FTM-PVCs表达减数分裂、卵细胞及透明带相关基因的情况。结果：1、含人卵泡液方案诱导人FTM-PVCs,细胞都会出现类卵丘-卵细胞样结构：人颗粒细胞与人FTM-PVCs共培养,也可出现类卵细胞样结构；2、人卵泡液诱导拟胚体和贴壁培养的人FTM-PVCs分化后,类卵细胞均表达卵细胞及透明带相关特异性标志物；3、人卵泡液诱导贴壁培养的人FTM-PVCs分化后,细胞中减数分裂标志物、卵细胞标志物和透明带标志物的mRNA表达增强；4、人卵泡液诱导拟胚体分化后,细胞培养液中E2浓度上升。结论：1、人FTM-PVCs为多能干细胞,与“精”功能相似2、首次证实人FTM-PVCs具有在体外分化为类卵细胞的潜力,诱导分化成的类卵细胞有分泌E2的能力第二部分：含补肾中药鼠血清对人FTM-PVCs体外分化为类卵细胞影响的研究方法：1、各类含补肾中药鼠血清的制备：在体重均一的情况下随机将50只大鼠分成5组：空白组、1号药组、2号药组、3号药组、5号药组,每组10只,雌雄各半。空白组大鼠灌服蒸馏水,1号、2号、3号和5号药组大鼠分别灌服对应号码的中药,给药剂量均为5.Og/kg,给药容量均为20ml/kg。分别于给药后30min、60min、90min眼底静脉丛取血,血液静置2小时后,3000rpm离心l0min,分离各组各不同时间段血清,将每组三个时间段的血清混匀,HPLC分离血清,过滤除菌备用。2、各类含补肾中药鼠血清对人FTM-PVCs向类卵细胞分化影响的研究：观察不同比例各类含补肾中药鼠血清对人FTM-PVCs向类卵细胞分化的影响：将人FTM-PVCs分为2%、5%、10%、20%含各类补肾中药鼠血清组、各类补肾中药鼠血清+卵泡液组、空白鼠血清组、空白鼠血清+卵泡液组,control组和Dositive control组,显微镜下观察每组细胞形态学变化并拍照。各组干细胞在不同条件下培养24天、31天后,细胞免疫组化检测卵细胞标志物GDF-9的表达；RT-PCR半定量检测2%含2号药鼠血清组细胞中减数分裂、卵细胞及透明带相关基因的表达情况；酶联免疫法检测该组细胞培养液中不同时间段的E2浓度。结果：1、各类含补肾中药鼠血清诱导人FTM-PVCs分化后,细胞形态学变化类似人卵泡液+FSH、LH+E2(?)日性对照组,均出现类卵细胞样结构；2、含补肾中药鼠血清诱导人FTM-PVCs分化后,细胞都能够表达卵细胞标志物GDF-9；3、含2号药鼠血清诱导人FTM-PVCs分化后,细胞表达的SCP3、DF-9、ZP-1、 ZP-2、ZP-3mRNA明显增强；细胞培养液中的E2浓度升高。结论：1、首次证实补肾中药具有在体外诱导人FTM-PVCs向类卵细胞分化的能力,与肾主生殖,为先天之本相关2、人FTM-PVCs可作为体外评价补肾中药对生殖细胞生长发育影响的新研究平台第三部分：含补肾中药鼠血清和人卵泡液诱导人FTM-PVCs体外分化为类卵细胞的机理研究方法：酶联免疫法检测人卵泡液和人卵泡液诱导人FTM-PVCs分化后细胞培养液中相关细胞因子和激素的浓度。酶联免疫法检测各类鼠血清和各类鼠血清诱导人FTM-PVCs分化后,细胞培养液中相关激素和细胞因子的浓度。液相色谱仪分析各类鼠血清成分。结果：1、人卵泡液中较文献报道人血清正常值明显升高的细胞因子有：F-T、VEGF;2、人卵泡液诱导贴壁培养的人FTM-PVCs分化后,细胞培养液中的VEGF、 SCP1、 SCP2、MPF浓度上升；3、各类含补肾中药鼠血清与空白鼠血清比较,血清中P、E2、FSH、LH、F-T、 MPF, SCF、EGF, IGF-1、TNF-α、 IL-1α的含量无明显差别,含2、3、5号药鼠血清与空白鼠血清比较VEGF水平有所升高；4、各类含补肾中药鼠血清与空白鼠血清比较,均有相同成分峰出现在16min～17min;含1号药鼠血清、含3号药鼠血清、含5号药鼠血清均有相同成份峰出现在2min～3min;5、含1号药鼠血清诱导人FTM-PVCs分化后,细胞培养液中的VEGF、SCP1、 SCP2、SCP3、 MPF浓度升高；含2号药鼠血清诱导人FTM-PVCs分化后,细胞培养液中的E2、SCP2、 MPF、 F-T升高；含3号药和5号药鼠血清诱导人FTM-PVCs分化后,细胞培养液中的SCP3、MPF升高。讨论与结论：1、人卵泡液所含VEGF, SCP1,SCP2和MPF可能在诱导人FTM-PVCs向类卵细胞分化的过程中起一定作用；2、含补肾中药鼠血清中的未知有效成分可能在诱导人FTM-PVCs向类卵细胞分化的过程中起某种间接作用。更多还原

【Abstract】 Objective:To investigate the effect of rat medicated sera prepared with kidney-invigorating Chinese herbal formulas on differentiation of human first trimester-derived perivascular cells (FTM-PVCs) into oocyte-like cells (OLCs) in vitro.First part:To investigate whether or not the human FTM-PVCs were able to differentiate in vitro into the OLCs and establish an in vitro research platform for the kidney-invigorating Chinese herbs.Methods:1. Methods for inducing human FTM-PVCs into OLCs in vitroThe human FTM-PV cells were first isolated and cultured according to the method published previously. After that, different differentiation protocols were investigated whether or not the cells were able to differentiate into OLCs. The protocols included1)25%human ovarian follicular fluid+FSH+LH,2).25%human ovarian follicular fluid+FSH+LH+E2,3).25%human ovarian follicular fluid+FSH+LH+E2+folic acid,4).25%human ovarian follicular fluid+FSH+LH+E2+IVM culturing medium and5). co-culture with human follicular granulosa cells or culture conditioned medium of human follicular granulosa cells. During the process of differentiation, morphological changes were compared under an inverted microscope among the various differentiation protocols. Expressions of specific markers for oocytes were determined by immunocytochemistry and estradiol concentrations in the culture media were assayed by using an estradiol ELISA.2. Establishment of the in vitro research platform for the kidney-invigorating Chinese herbsAfter comparisons, culture with25%human ovarian follicular fluid+FSH+LH+E2was selected as a standard differentiation protocol. Morphologic observation, detection of specific biomarkers for oocytes such as SCP3, GDF-9, ZP1, ZP2and ZP3at transcription and translation levels, as well as estradiol production were all included into the platform.Results: 1. The OLCs and/or cumulus-oocyte-like complex (COCs) in morphology could be found after24days of culturing the FTM-PVCs in human ovarian follicular fluid plus LH, FSH and E2or co-culturing with human granulosa cells.2. The OLCs could express SCP3, GDF-9, ZP1, ZP2and ZP3at transcription and translation levels.3. The estradiol concentrations in the culture media of the OLCs were significantly higher than that in the culture media of the undifferentiated FTM-PVCs.Conclusions:1. Human FTM-PVCs have a potential ability to differentiate into the OLCs in vitro in the culture with human follicular fluid or co-culture with human granulose cells. To our knowledge, this is the first report of differentiation of OLCs derived from non-embryonic stem cells.2. The characteristics of FTM-PVCs are similar to "essence" as defined by theories of Traditional Chinese Medicine. Therefore, the differentiation process from FTM-PVCs to OLCs could be used as a research platform for investigation of Chinese herbal function.Second part:To investigate the effect of rat medicated sera prepared with kidney-invigorating Chinese herbal formulas on the differentiation process from human FTM-PVCs to OLCs in vitroMethods:1. The preparation of rat medicated sera with invigorating kidney herbsFifty rats were randomly divided into5groups:blank group,1#drug group,2#drug group,3#drug group and5#drug group,10in each group which included5males and5females. The rats in blank group were drenched with distilled water while the rats in1#,2#,3#or5#groups were drenched with corresponding herbal formula decoctions of20ml/kg body weight (equal to5.0g drug/kg body weight)._After90min of drug administrations, rat blood samples were collected from eye-ground veins, and the medicated or blank sera were obtained by centrifugation at3000rpm for10min. The sera were purified with HPLC and filtered to sterile.2. The investigation of the effect of the rat medicated sera on the differentiation process from human FTM-PVCs to OLCs in vitroThe human FTM-PVCs were cultured with2%,5%,10%or20%of the various rat medicated serum. At the same time, the cells were also treated with the rat blank serum as a negative control, while the human follicular fluid as a positive control. Moreover, the rat medicated sera plus human follicular fluid were used to observe whether or not there was a synergistic effect of the rat medicated sera and human follicular fluid. The morphological changes for each group was observed under an inverted microscope. On the24th or31st day after interventions, the expression of GDF-9was examined with immunocytochemistry assay. For the group with2%of the rat medicated serum, mRNA levels of SCP3, GDF-9, ZP1, ZP2and ZP3were evaluated with a semi-quantitative RT-PCR assay. The E2productions in culture medium were measured with an ELISA assay.Results:1. The OLCs in morphology could be found in cultures with the#2,#3and#5rat medicated serum that were similar to the positive controls while no significant changes in cell morphology was found in the negative controls. Moreover, no a synergistic effect of the rat medicated serum and human follicular fluid can be detected.2. After the intervention with the rat medicated sera, the differentiated OLCs could express GDF-9protein, and mRNA levels of SCP3, GDF-9, ZP-1, ZP-2and ZP-3were significantly higher than that in the negative controls.3. E2production in culture media with the rat medicated sera was significantly higher than that in culture media with the rat blank sera.Conclusions:1. The rat medicated sera prepared with invigorating kidney herbs has the ability to induce human FTM-PVCs into OLCs in vitro. The findings may demonstrated the TCM theory of "Kidney controls reproduction and is congenital foundation" from the view of modern Western medicine.2. The differentiation process from the human FTM-PVCs differentiation into the OLCs can be used as a new platform to evaluate the effect of invigorating kidney herbs on the growth and development of germ cells in vitro.Third part:To investigate possible mechanisms underlying the effect of the rats medicated seraMethods:Components of the various rat medicated sera were analyzed with HPLC. Concentrations of various cytokines and hormones in the rat medicated sera or human follicular fluid as well as differentiation culture media with the rat medicated sera or human follicular fluid were compared by using a number of specific ELISA kits, respectively.Results:1. There were different peaks appeared at2-3min or16-17min in1#,3#and5#rat drug serum from the rat blank serum on the HPLC profile.2. In the human ovarian follicular fluid, there were a number of molecules associated with germ cell formation/maturation such as VEGF, SCP1, SCP2, SCP3, MPF and SCF.3. After inducing FTM-PVCs into the OLCs by using human ovarian follicular fluid, the concentrations of VEGF, SCP1, SCP2, MPF in the culture medium of OLCs were significantly increased when compared to the culture medium of undifferentiated cells.4. The levels of P, E2, FSH, LH, F-T, MPF, SCF, EGF, IGF-1, TNF-a, IL-la in the rat medicated sera were not significantly different from that in the rat blank sera. However, the VEGF levels in the2#,3#and5#rat medicated were significantly higher that in rat blank serum.5. Compared to the negative controls, the concentrations of VEGF, SCP1, SCP2, SCP3and MPF in culture medium with the1#rat medicated serum were increased while the levels of E2, SCP2, MPF and F-T in culture medium with2#rat medicated serum and the levels of SCP3and MPF in culture medium with3#and5#rat medicated sera were elevated.Conclusions:1. From the results, it suggests that VEGF, SCP1, SCP2and MPF in human follicular fluid may play an important role in the differentiation of the FTM-PVCs into the OLCs since levels of these molecules in the culture medium of the OLCs were significantly increased compared to that of the undifferentiated cells.2. The possible mechanism underlying the effects of rat medicated sera may be similar to that of human follicular fluid by increase of production of VEGF, SCP1, SCP2, SCP3, MPF and free testosterone from the differentiated cells. However, it may be induced by some unknown active components in the rat medicated sera rather than directly by these molecules since the levels of molecules of rat medicated sera were not different from the blank serum.更多还原