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
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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。
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 vitro
The 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 herbs
After 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 vitro
Methods:
1. The preparation of rat medicated sera with invigorating kidney herbs
Fifty 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 vitro
The 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 sera
Methods:
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.
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。
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 vitro
The 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 herbs
After 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 vitro
Methods:
1. The preparation of rat medicated sera with invigorating kidney herbs
Fifty 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 vitro
The 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 sera
Methods:
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.
引文
[1]沈自尹.“肾”研究成果对中西医结合研究思路的启示.中国中医药报,2010,5(1)：1
[2]张进,徐志伟,杜少辉,等.“精”学说与干细胞辨识.中医药学刊[J].2004,22(7)：1198-1200
[3]徐志伟,张进,张明.从心肾相关探讨干细胞移植治疗冠心病心衰的中医理论内涵[J].云南中医学院学报,2009,(5)：1-4
[4]何绍清,罗振宇,刘秋英,等.人脐带间充质于细胞分离培养及向脂肪与成骨细胞的分化[J].中国组织工程研究与临床康复,2010,14(14)：2492-2496
[5]柳菁,许超,宇丽,等.两种培养体系条件下人脐带间充质干细胞向软骨细胞的分化[J].中国组织工程研究,2012,16(41)：7625-7630
[6]朱华民,张洹肝细胞生长因子与成纤维生长因子联合诱导人脐带间充质干细胞向肝样细胞的分化[J].中国组织工程研究与临床康复.2009,13(45)：8811-8816
[7]林晓波,何红燕,罗敏洁,等.人脐带间充质干细胞向心肌样细胞分化的研究[J].实用儿科临床杂志,2007,22(13)：971-973,977
[8]王爱红,何红燕,罗敏洁,等.人脐带间充质干细胞向胰岛样细胞的分化[J].中国组织工程研究与临床康复,2008,12(21)：4102-4106
[9]Ma L, Feng XY, Cui BL, et al. Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells [J]. Chinese Medical Journal,2005,118(23):1987-1993
[10]孙国栋,李志忠.人脐带间充质干细胞在组织工程中的研究进展[J].现代生物医学进展,2009,9(12)：2389-2390
[11]Hong SH, Maghen L, Kenigsberg S, et al. Ontogeny of human umbilical cord perivascular cells:molecular and fate potential changes during gestation[J]. Stem Cells Dev.2013,5(14)
[12]Hubner K, Fuhrmann G, Christenson LK, et al. Derivation of oocytes from mouse embryonic stem cells[J]. Science,2003,300(5623):1251-1256
[13]Lacham. Kaplan 0, Chy H, Trounson A. Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes[J]. Stem Cells,2005; 24(2):266-273
[14]Novak I, Lightfoot D A, Wang H, et al. Mouse embryonic stem cells form follicle-like ovarian structures but do not progress through meiosis[J]. Stem Cells,2006,24:1931-1936
[15]Clark AT, BodnarMS, FoxM, et al. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro [J].Hum MolGenet,2004,13:727-739.
[16]李彩霞.人脐带基质干细胞体外向雌性生殖细胞分化的潜能[J].中国组织工程研究与临床康复,2010,14(40)：7583-7587
[17]吕品,何玲,柴惠霞.类胚体诱导脐带间充质干细胞向生殖细胞的分化[J].中国组织工程研究,2012,16(23)：4217-4221
[18]郑洪新,王拥军,李佳,等.“肾藏精”与干细胞及其微环境及NEI网络动态平衡关系[J].中华中医药杂志.2012,27(9)：2267-2270
[19]Dong Ryul Lee, Kye-Seong Kim, Yun Hee Yang, et al. Isolation of male germ stem cell-like cells from testicular tissue of non-obstructive azoospermic patients and differentiation into haploid male germ cells in vitro[J]. Human Reproduction,2006,21(2):471-476
[20]S. Danner, J. Kajahn, C. Geismann, et al. Derivation of oocyte-like cells from a clonal pancreatic stem cell line.Molecular Human Reproduction,2007,13(1):11-20
[21]Lisa M. Salvador, Celso P. Silva, Igor Kostetskii,et al.The Promoter of the Oocyte-Specific Gene, Gdf9, is Active in a Population of Cultured Mouse Embryonic Stem Cells with an Oocyte-Like Phenotype[J]. Methods,2008,45(2):172-181
[22]Paul W, Dyce, Wei Shen, et al. Analysis of Oocyte-Like Cells Differentiated from Porcine Fetal Skin-Derived Stem Cells [J].Stem Cells and Develpement, 2011,20(5):809-819
[23]Looijenga LH, StoopH, de Leeuw H P, et al. POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors [J].Cancer Res,2003,63: 2244-2250
[24]Bhattacharya B, Miura T, B randenberger R,et al.Gene expression in hum an embryonic stem cell lines:unique molecular signature[J]. Blood,2004,103 (8) 2956-2964
[25]Nichols J, BZevnik, K Anastassiadis, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4[J].Cell,1998,95(3)-.379-391
[26]Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells [J]. Cell,2003, 113(5):643-655
[27]Mitsui K, Tokuzawa Y, Itoh H, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and cells [J]. Cell,2003, 113(5):631-642
[28]Clark AT, BodnarMS, FoxM, et al. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro [J].Hum MolGenet,2004,13:727-739
[29]SaitouM, Barton SC, SuraniMA, et al. A molecular programme for the specification of germ cell fate in mice [J]. Nature,2002,418:293-300
[30]Clark AT, Rodriguez RT, Bodnar MS, et al. Human STELLAR, NANOG, and GDF3 genes are Expressed in pluripotent cells andmap to Chromosome 12p13, a hotspot for teratocarcinoma [J].Stem Cells,2004,22:169-179
[31]Lange UC, Saitou M, Western PS, etal. The fragilis inter-feron-inducible gene family of transmembrane proteins is associated with germ cell specification in mice [J]. BMC Dev Biol,2003,3:1-11
[32]Teng YN, Lin YM, Sun HF, et al. Association of DAZL haplotypes with spermatogenic failure in infertile men [J].Fertil Steril,2006,86(1)-.129-135
[33]Xu EY, Moore FL, Reijo RA. From the Cover:A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans [J]. PNAS,2001,98:7414-7419.
[34]Kehkooi Kee, Vanessa T Angeles, et al. Human DAZL, DAZ and BOULE genes modulate primordial germ cell and haploid gamete formation [J]. Nature,2009, 462(7270):222-225
[35]Schrans-Stassen BH, Saunders PT, Cooke HJ, et al. Nature of the spermatogenic arrest in DAZL-/-mice[J]. Biol Reprod,2001,65(3):771-776
[36]Alberts B, Bray D, Lewis J. Molecular Biology of the Cell (4th Ed) [M]. New York:Publishing Inc,2002,1135-1137
[37]Zickler D, Kleckner N. Meiotic chromosomes:integrating structure and function[J]. Annu Rev Genet,1999,33:603-754
[38]Heyting C. Synaptonemal complex:structure and function[J]. Curr Opin Cell Bio,1996,8(3):389-396
[39]Yuan L, Liu JG, Zhao J, et al. Themurine SCP3 gene is required for synaptonem alcomplex assembly, chromosome synapsis, and male fertility [J]. MolCell 2000,5: 73-83
[40]Yuan L, Liu JG, Hoja MR, et al. Female germ cell aneuploidy and embryo death in mice lacking the meiosis-specific protein SCP3[J]. Science,2002, 296:1115-1118
[41]Pelttari J, Hoja MR, Yuan L, et al. A meiotic chromosomal core consisting of cohesion complex proteins recruits DNA recombination proteins and promotes synapsis in the absence of an axial element in mammalian meiotic cells[J]. Mol Cell Biol, 2001,21:5667-5677
[42]McPherronn A C, Lee S J. GDF-3 and GDF-9:two new members of the transforming growth factor beta superfamily containing a novel pattern of cysteines[J]. Biol Chen,1993,268(5):3444-3449
[43]McGrath SA, Esquela AF, Lee SJ. Oocyte-specific expression of growth/differentiation factor-9 [J]. Mol Endocrinol,1995,9(1):131-136
[44]Julia A. Elvin, Amander T. Clark, Pei Wang, et al.Paracrine actions of growth differentiation factor-9 in the mammalian overy [J]. Mol Endocrinol,1999,13(6):1035-1048
[45]Li HK, Kuo TY, Yang HS, et al. Differential gene expression of bone morphogenetic protein 15 and growth differentiation factor 9 during in vitro maturation of porcine oocytes and early embryos [J]. Animal Reproduction Science,2008,103(3-4):312-322
[46]Castrillon DH, Quade BJ, Wang TY, et al. The human VASA gene is specifically expressed in the germ cell line-age [J]. PNAS,2000,97:9585-9590.
[47]Liu C, Litscher E S, Mortillo S, et al. Targeted disruption of the mZP3 gene results in production of eggs lacking a zona pellucida and infertility in female mice [J]. Proc Natl Acad Sci USA,1996,93:5431-5436
[48]David G. Mottershead, Minna M. Pulkki, Pranuthi Muggalla, et al. Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells[J]. Molecular and Cellular Endocrinology,2008,283 (1-2):58-67
[49]Yamamoto, N, Christenson LK, McAllister JM, et al, Growth Differentiation Factor-9 Inhibits 3'5'-Adenosine Monophosphate-Stimulated Steroidogenesis in Human Granulosa and Theca Cells [J]. The Journal of Clinical Endocrinology& Metabolism,2002,87(6):2849-2856
[50]Prasad S V, Skinner S M, Carinoc C, et al. Structure and function of the proteins of the mammalian zona pellucida [J]. Cells Tissues Organs,2000,166:148-164
[51]谢妍,乜春城,曹佐武.小鼠ZP2mRNA在卵泡颗粒细胞层的表达[J].暨南大学学报(医学版),2010,31(2)：116-119
[52]GOOK D A, EDGAR D H, BORG J, et al. Detection of zona pellucida prote ins during human fo llicu logenesis [J]. Hum Reprod,2008,23(2):394-402
[53]杨宏敏,杜惠兰补肾调经方、逍遥丸人含药血清对人卵巢颗粒细胞E2、P和FSHR影响的比较研究,第十次全国中医妇科学术大会论文集,2010
[54]夏天,韩冰.补肾调冲方含药血清对大鼠卵巢颗粒细胞增殖、激素分泌和mRNA表达的影响[J].现代药物与临床,2011,26(5)：384-388
[55]孙晓峰,尤昭玲,刘丹卓.补肾活血方对PCO小鼠未成熟卵母细胞体外核成熟的影响[J].湖南中医药大学学报,2011,31(3)：27-30
[56]邢长英,张德祥,滕银成,等.补肾益气方激活哺乳动物雷帕霉素靶分子(mTOR)通路影响人早孕细胞滋养层细胞生长[J].生殖与避孕,2009,29(9)：584-588
[57]王海燕,归绥琪,徐丛剑,等.补肾益气方对正常早孕期人细胞滋养层细胞生物学行为的影响[J].中国中西医结合杂志,2004,24(6)：525-528
[58]胡兵,安红梅,史云峰,等.补肾阴对胚胎干细胞关键基因功能的影响[J].中国中医基础医学杂志,2007,13(5)：365-367
[59]安红梅,胡兵,史云峰,等.补肾阴对胚胎干细胞活动的影响[J].中国中医药信息杂志,2007,14(6)：24-26
[60]安红梅,胡兵,史云峰,等.补肾阳中药对小鼠胚胎干细胞活动及相关基因表达的影响[J].中华中医药杂志(原中国医药学报),2008,23(9)：774-776
[61]徐凌霄,王芳,郭敦明,等.左归丸对间充质干细胞向软骨细胞分化过程中Ⅱ型胶原及蛋白多糖基因表达的影响[J].中国中西医结合杂志,2011,31(12)：1662-1668
[62]谭峰,樊巧玲,王明艳等.左归丸对SD大鼠骨髓间充质干细胞增殖的影响[J].中国实验方剂学杂志,2011,17(13)：145-148
[63]Jiang XH, Yie SM, Zhen X, et al. Effect of You Gui Wan on mouse sperm fertilising ability in vivo and in vitro[J]. Andrologia.2013 Mar 6
[64]刘永琦,蔡玲,颜春鲁,等.补肾中药对大鼠BMSCs分泌NGF β、BDNF、VEGF功能的影响[J].免疫学杂志,2010,26(10)：850-853
[65]黄勇,黄秀深,胡一梅,等.“左归丸”配伍规律对骨髓源成体干细胞定向分化的调控作用[J].成都中医药大学学报,2010,33(1)：48-52
[66]胡黎平,涂平生,谢富康,等.补肾方剂对大鼠骨髓基质干细胞增殖与分化的影响[J].中国药物与临床,2007,7(5)：329-331
[67]陈东风,杜少辉,李伊为,等.龟板对局灶性脑缺血后神经干细胞的作用[J].广州中医药大学学报,2001,18(4)：328-331
[68]张进,徐志伟.补肾法诱导间充质干细胞向神经方向分化研究[J].现代医院,2004,4(9)：15-17
[69]曾建春,樊粤光,刘建仁,等.杜仲含药血清诱导骨髓间充质干细胞定向分化的实验研究[J].时珍国医国药,2009,20(9)：2136-2138
[70]张贤,蔡建平,张艳红.杜仲诱导大鼠间充质干细胞成骨分化中成骨与成脂相关转录因子的表达[J].中国组织工程研究与临床康复,2010,14(19)：3523-3526
[71]黄勇,吴建军,刘凯,等.熟地含药血清对骨髓间质干细胞向神经细胞分化的影响[JJ.中国老年学杂志,2011,31：3979-3981
[72]张永占,刘,占彦.补肾中药对女性生殖系统功能的影响[J].河南中医学院学报,2006,21(1)：85-88
[73]杨丽芸,杜惠兰,白静,等.补肾法、疏肝法对超促排卵小鼠卵母细胞数量及GDF-9表达的影响[J].中医杂志,2013,54(7)：597-604
[74]Karahuseyinoglu S, Cinar 0, Kilic E, et al. Biology of stem cells in human umbilical cord stroma:in situ and in vitro surveys[J]. Stem Cells,2007,25(2):319-331
[75]Hou Ke-dong, Xu Wen-jing, Zhang Li,et al.To study chondrogenic induction of mesenchymal stem cells derived from Wharton jelly of human umbilical cord[J]. Chinese Journal of Orthopaedics,2007,27 (12):930-935
[76]陈军,刘玉侠,姜文华,等.体外诱导人脐带间充质干细胞向肝样细胞的分化[J].中国组织工程研究与临床康复,2010,14(49)：9257-9261
[77]Can A,Karahuseyinoglu S. Concise review:human umbilical cord stroma with regard to the source of fetus-derived stem cells[J]. Stem Cells,2007,25(11).2886-2895
[78]Mitchell K E,Weiss M L.Mitchell B M, et al. Matrix cells from Whar-ton" s jelly form neurons and glia[J].Stem Cells,2003,21(1):50-60
[79]Dyce PW, Liu J, Tayade C, et al. In vitro and in vivo germ line potential of stem cells derived from newborn mouse skin[J]. PLoS One.2011,6(5):1-14
[80]Dyce PW, Wen L, Li J. In vitro germline potential of stem cells derived from fetal porcine skin[J]. Nature Cell Biology,2006,8(4):384-90
[81]Danner S,Kajahn J, Geismann C, et al.Derivation of oocyte-like cells from a clonal pancreatic stem cell line[J].Mol Hum Reprod,2007,13(1):11-20.
[82]Johnson J, Bagley J, Skaznik-Wikiel M, et al. Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood[J]. Cell,2005, 122(2):303-315
[83]常秀峰,杜惠兰,高星,等.补肾调经方对输卵管性不孕症患者体外受精-胚胎移植超排卵周期GDF-9的影响[J].中国中西医结合杂志,2011,31(6)：780-783
[84]饶研文,卢雪芳,罗明,等.71例克氏综合征患者血清性激素和游离睾酮检测分析[J].现代诊断与治疗,2009,20(3)：174
[85]张四林,杨菁,尹太郎,等.卵泡液中VEGF水平对卵母细胞成熟及早期胚胎发育的影响[J].武汉大学学报(医学版),2008,29(1)：60-62
[86]张雪青,张李雅,徐春泉,等.少精子症患者血清和精液中游离睾酮和双氢睾酮水平的 测定及临床意义[J].中国优生与遗传杂志,2007,15(2)：98-104
[87]牛小平,韩真,何池义,等.EGF、VEGF在常见消化道肿瘤患者血清中的表达及意义[J].现代肿瘤医学,2007,15(5)：668-669
[88]Balasch J,Guimera M, Pasarell OM, et al. Adrenomedullin and vascular endothelial growth factorproduction by follicular fluid macrophages and granulose cells [J]. Hum Reprod,2004,19(4):808-814
[89]Luo H, Kimura K, Aoki M,, et al. Effect of vascular endothelial growth factor on maturation, fertilization and developmental competence of bovine oocytes [J]. Veterinary Medical Science,2002,9:803-806
[90]Hailing Luo, Kimura K, M, Aoki, et al. Vascular Endothelial Growth Factor (VEGF) Promotes the Early Development of Bovine Embryo in the Presence of Cumulus Cells [J]. Veterinary Medical Science,2002,11:967-971
[91]董晓晨,王稳,罗永,等.血管内皮生长因子对猪卵母细胞体外成熟的影响研究[J].中国农学通报2012,28(23)：50-55
[92]夏蕾,张岭,吴坤.卵母细胞成熟过程MPF的调控和MPF的作用底物[J].疾病控制杂志,2007,11(6)：614-617
[93]A Picard, S Galas, G Peaucellier, et al. Newly assembled cyclin B-cdc2 kinase is required to suppress DNA replication between meiosis I and meiosis II in starfish oocytes[J].EMBO,1996,15(14):3590-3598.
[94]彭佑共,杨罗艳.Hela细胞MPF对未成熟卵母细胞的体外促成熟作用分析[J].医学临床研究,2008,25(9)：1624-1627
[95]沈自尹,黄建华.从淫羊藿激活内源性干细胞探讨“肾藏精”的科学涵义[J].中医杂志,2010,51(1)：8-10
[96]Yong-jun Wang, Ruth M Belf lower, Yu-Feng Dong, et al. Runxl/AML1/Cbf a 2 mediates onset of mesenchymal cell differentiation toward chondrogenesis[J]. Journal of Bone and Mineral Research,2005,20(9):1624-1636
[97]Do Y Soung, Yufeng Dong, Yong-Jun Wang, et al. Runx3/AML2/Cbf a 3 regulates early and late chondrocyte differentiation [J]. Journal of Bone and Mineral Research,2007, 22(8):1260-1270
[98]Yvonne A R White, Dori C Woods, Yasushi Takai, et al. Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women [J]. Nature Medicine,2012,18:413-421
[1]Hubner K, Fuhrmann G, Christenson LK, et al. Derivation of oocytes from mouse embryonic stem cells[J]. Science,2003,300(5623):1251-1256
[2]Lacham. Kaplan 0, ChyH, Trounson A. Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes[J]. Stem Cells,2005; 24(2):266-273
[3]Novak I, Lightfoot D A, Wang H, et al. Mouse embryonic stem cells form follicle-like ovarian structures but do not progress through meiosis[J], Stem Cells,2006,24: 1931-1936
[4]Clark AT, BodnarMS, FoxM, et al. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro [J].Hum MolGenet,2004,13:727-739
[5]Johnson J, Bagley J, Skaznik-Wikiel M, et al. Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood[J]. Cell,2005, 122(2):303-315
[6]Dyce PW, Wen L, Li J. In vitro germline potential of stem cells derived from fetal porcine skin[J]. Nature Cell Biology,2006,8(4):384-90
[7]Danner S,Kajahn J,Geismann C, et al. Derivation of oocyte-like cells from a clonal pancreatic stem cell line[J].Mol Hum Reprod,2007,13(1):11-20.
[8]Dyce PW, Liu J, Tayade C, et al. In vitro and in vivo germ line potential of stem cells derived from newborn mouse skin[J].PLoS One,2011,6(5):1-14
[9]杨文成,董有海.人脐带间充质干细胞的研究进展[J].组织工程与重建外科杂志,2010,6(5)：292-294
[10]孙国栋,李志忠.人脐带间充质干细胞在组织工程中的研究进展[J].现代生物医学进展,2009,9(12)：2389-2390
[11]Mareschi K, I Ferrero, D Rustichelli, et al. Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow[J]. Cell Biochem, 2006,97(4):744-754.
[12]Choumerianou DM, G Martimianaki, E Stiakaki, et al. Comparative study of stemness characteristics of mesenchymal cells from bone marrow of children and adults[J]. Cytotherapy,2010,12:881-887.
[13]李彩霞.人脐带基质干细胞体外向雌性生殖细胞分化的潜能[J].中国组织工程研究与临床康复,2010,14(40)：7583-7587
[14]吕品,何玲,柴惠霞.类胚体诱导脐带间充质干细胞向生殖细胞的分化[J].中国组织 工程研究,2012,16(23)：4217-4221
[15]Hong SH, Maghen L, Kenigsberg S, et al. Ontogeny of human umbilical cord peri vascular cells:molecular and fate potential changes during gestation. Stem Cells Dev.2013,5(14)
[2]张进,徐志伟,杜少辉,等.“精”学说与干细胞辨识.中医药学刊[J].2004,22(7)：1198-1200
[3]徐志伟,张进,张明.从心肾相关探讨干细胞移植治疗冠心病心衰的中医理论内涵[J].云南中医学院学报,2009,(5)：1-4
[4]何绍清,罗振宇,刘秋英,等.人脐带间充质于细胞分离培养及向脂肪与成骨细胞的分化[J].中国组织工程研究与临床康复,2010,14(14)：2492-2496
[5]柳菁,许超,宇丽,等.两种培养体系条件下人脐带间充质干细胞向软骨细胞的分化[J].中国组织工程研究,2012,16(41)：7625-7630
[6]朱华民,张洹肝细胞生长因子与成纤维生长因子联合诱导人脐带间充质干细胞向肝样细胞的分化[J].中国组织工程研究与临床康复.2009,13(45)：8811-8816
[7]林晓波,何红燕,罗敏洁,等.人脐带间充质干细胞向心肌样细胞分化的研究[J].实用儿科临床杂志,2007,22(13)：971-973,977
[8]王爱红,何红燕,罗敏洁,等.人脐带间充质干细胞向胰岛样细胞的分化[J].中国组织工程研究与临床康复,2008,12(21)：4102-4106
[9]Ma L, Feng XY, Cui BL, et al. Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells [J]. Chinese Medical Journal,2005,118(23):1987-1993
[10]孙国栋,李志忠.人脐带间充质干细胞在组织工程中的研究进展[J].现代生物医学进展,2009,9(12)：2389-2390
[11]Hong SH, Maghen L, Kenigsberg S, et al. Ontogeny of human umbilical cord perivascular cells:molecular and fate potential changes during gestation[J]. Stem Cells Dev.2013,5(14)
[12]Hubner K, Fuhrmann G, Christenson LK, et al. Derivation of oocytes from mouse embryonic stem cells[J]. Science,2003,300(5623):1251-1256
[13]Lacham. Kaplan 0, Chy H, Trounson A. Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes[J]. Stem Cells,2005; 24(2):266-273
[14]Novak I, Lightfoot D A, Wang H, et al. Mouse embryonic stem cells form follicle-like ovarian structures but do not progress through meiosis[J]. Stem Cells,2006,24:1931-1936
[15]Clark AT, BodnarMS, FoxM, et al. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro [J].Hum MolGenet,2004,13:727-739.
[16]李彩霞.人脐带基质干细胞体外向雌性生殖细胞分化的潜能[J].中国组织工程研究与临床康复,2010,14(40)：7583-7587
[17]吕品,何玲,柴惠霞.类胚体诱导脐带间充质干细胞向生殖细胞的分化[J].中国组织工程研究,2012,16(23)：4217-4221
[18]郑洪新,王拥军,李佳,等.“肾藏精”与干细胞及其微环境及NEI网络动态平衡关系[J].中华中医药杂志.2012,27(9)：2267-2270
[19]Dong Ryul Lee, Kye-Seong Kim, Yun Hee Yang, et al. Isolation of male germ stem cell-like cells from testicular tissue of non-obstructive azoospermic patients and differentiation into haploid male germ cells in vitro[J]. Human Reproduction,2006,21(2):471-476
[20]S. Danner, J. Kajahn, C. Geismann, et al. Derivation of oocyte-like cells from a clonal pancreatic stem cell line.Molecular Human Reproduction,2007,13(1):11-20
[21]Lisa M. Salvador, Celso P. Silva, Igor Kostetskii,et al.The Promoter of the Oocyte-Specific Gene, Gdf9, is Active in a Population of Cultured Mouse Embryonic Stem Cells with an Oocyte-Like Phenotype[J]. Methods,2008,45(2):172-181
[22]Paul W, Dyce, Wei Shen, et al. Analysis of Oocyte-Like Cells Differentiated from Porcine Fetal Skin-Derived Stem Cells [J].Stem Cells and Develpement, 2011,20(5):809-819
[23]Looijenga LH, StoopH, de Leeuw H P, et al. POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors [J].Cancer Res,2003,63: 2244-2250
[24]Bhattacharya B, Miura T, B randenberger R,et al.Gene expression in hum an embryonic stem cell lines:unique molecular signature[J]. Blood,2004,103 (8) 2956-2964
[25]Nichols J, BZevnik, K Anastassiadis, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4[J].Cell,1998,95(3)-.379-391
[26]Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells [J]. Cell,2003, 113(5):643-655
[27]Mitsui K, Tokuzawa Y, Itoh H, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and cells [J]. Cell,2003, 113(5):631-642
[28]Clark AT, BodnarMS, FoxM, et al. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro [J].Hum MolGenet,2004,13:727-739
[29]SaitouM, Barton SC, SuraniMA, et al. A molecular programme for the specification of germ cell fate in mice [J]. Nature,2002,418:293-300
[30]Clark AT, Rodriguez RT, Bodnar MS, et al. Human STELLAR, NANOG, and GDF3 genes are Expressed in pluripotent cells andmap to Chromosome 12p13, a hotspot for teratocarcinoma [J].Stem Cells,2004,22:169-179
[31]Lange UC, Saitou M, Western PS, etal. The fragilis inter-feron-inducible gene family of transmembrane proteins is associated with germ cell specification in mice [J]. BMC Dev Biol,2003,3:1-11
[32]Teng YN, Lin YM, Sun HF, et al. Association of DAZL haplotypes with spermatogenic failure in infertile men [J].Fertil Steril,2006,86(1)-.129-135
[33]Xu EY, Moore FL, Reijo RA. From the Cover:A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans [J]. PNAS,2001,98:7414-7419.
[34]Kehkooi Kee, Vanessa T Angeles, et al. Human DAZL, DAZ and BOULE genes modulate primordial germ cell and haploid gamete formation [J]. Nature,2009, 462(7270):222-225
[35]Schrans-Stassen BH, Saunders PT, Cooke HJ, et al. Nature of the spermatogenic arrest in DAZL-/-mice[J]. Biol Reprod,2001,65(3):771-776
[36]Alberts B, Bray D, Lewis J. Molecular Biology of the Cell (4th Ed) [M]. New York:Publishing Inc,2002,1135-1137
[37]Zickler D, Kleckner N. Meiotic chromosomes:integrating structure and function[J]. Annu Rev Genet,1999,33:603-754
[38]Heyting C. Synaptonemal complex:structure and function[J]. Curr Opin Cell Bio,1996,8(3):389-396
[39]Yuan L, Liu JG, Zhao J, et al. Themurine SCP3 gene is required for synaptonem alcomplex assembly, chromosome synapsis, and male fertility [J]. MolCell 2000,5: 73-83
[40]Yuan L, Liu JG, Hoja MR, et al. Female germ cell aneuploidy and embryo death in mice lacking the meiosis-specific protein SCP3[J]. Science,2002, 296:1115-1118
[41]Pelttari J, Hoja MR, Yuan L, et al. A meiotic chromosomal core consisting of cohesion complex proteins recruits DNA recombination proteins and promotes synapsis in the absence of an axial element in mammalian meiotic cells[J]. Mol Cell Biol, 2001,21:5667-5677
[42]McPherronn A C, Lee S J. GDF-3 and GDF-9:two new members of the transforming growth factor beta superfamily containing a novel pattern of cysteines[J]. Biol Chen,1993,268(5):3444-3449
[43]McGrath SA, Esquela AF, Lee SJ. Oocyte-specific expression of growth/differentiation factor-9 [J]. Mol Endocrinol,1995,9(1):131-136
[44]Julia A. Elvin, Amander T. Clark, Pei Wang, et al.Paracrine actions of growth differentiation factor-9 in the mammalian overy [J]. Mol Endocrinol,1999,13(6):1035-1048
[45]Li HK, Kuo TY, Yang HS, et al. Differential gene expression of bone morphogenetic protein 15 and growth differentiation factor 9 during in vitro maturation of porcine oocytes and early embryos [J]. Animal Reproduction Science,2008,103(3-4):312-322
[46]Castrillon DH, Quade BJ, Wang TY, et al. The human VASA gene is specifically expressed in the germ cell line-age [J]. PNAS,2000,97:9585-9590.
[47]Liu C, Litscher E S, Mortillo S, et al. Targeted disruption of the mZP3 gene results in production of eggs lacking a zona pellucida and infertility in female mice [J]. Proc Natl Acad Sci USA,1996,93:5431-5436
[48]David G. Mottershead, Minna M. Pulkki, Pranuthi Muggalla, et al. Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells[J]. Molecular and Cellular Endocrinology,2008,283 (1-2):58-67
[49]Yamamoto, N, Christenson LK, McAllister JM, et al, Growth Differentiation Factor-9 Inhibits 3'5'-Adenosine Monophosphate-Stimulated Steroidogenesis in Human Granulosa and Theca Cells [J]. The Journal of Clinical Endocrinology& Metabolism,2002,87(6):2849-2856
[50]Prasad S V, Skinner S M, Carinoc C, et al. Structure and function of the proteins of the mammalian zona pellucida [J]. Cells Tissues Organs,2000,166:148-164
[51]谢妍,乜春城,曹佐武.小鼠ZP2mRNA在卵泡颗粒细胞层的表达[J].暨南大学学报(医学版),2010,31(2)：116-119
[52]GOOK D A, EDGAR D H, BORG J, et al. Detection of zona pellucida prote ins during human fo llicu logenesis [J]. Hum Reprod,2008,23(2):394-402
[53]杨宏敏,杜惠兰补肾调经方、逍遥丸人含药血清对人卵巢颗粒细胞E2、P和FSHR影响的比较研究,第十次全国中医妇科学术大会论文集,2010
[54]夏天,韩冰.补肾调冲方含药血清对大鼠卵巢颗粒细胞增殖、激素分泌和mRNA表达的影响[J].现代药物与临床,2011,26(5)：384-388
[55]孙晓峰,尤昭玲,刘丹卓.补肾活血方对PCO小鼠未成熟卵母细胞体外核成熟的影响[J].湖南中医药大学学报,2011,31(3)：27-30
[56]邢长英,张德祥,滕银成,等.补肾益气方激活哺乳动物雷帕霉素靶分子(mTOR)通路影响人早孕细胞滋养层细胞生长[J].生殖与避孕,2009,29(9)：584-588
[57]王海燕,归绥琪,徐丛剑,等.补肾益气方对正常早孕期人细胞滋养层细胞生物学行为的影响[J].中国中西医结合杂志,2004,24(6)：525-528
[58]胡兵,安红梅,史云峰,等.补肾阴对胚胎干细胞关键基因功能的影响[J].中国中医基础医学杂志,2007,13(5)：365-367
[59]安红梅,胡兵,史云峰,等.补肾阴对胚胎干细胞活动的影响[J].中国中医药信息杂志,2007,14(6)：24-26
[60]安红梅,胡兵,史云峰,等.补肾阳中药对小鼠胚胎干细胞活动及相关基因表达的影响[J].中华中医药杂志(原中国医药学报),2008,23(9)：774-776
[61]徐凌霄,王芳,郭敦明,等.左归丸对间充质干细胞向软骨细胞分化过程中Ⅱ型胶原及蛋白多糖基因表达的影响[J].中国中西医结合杂志,2011,31(12)：1662-1668
[62]谭峰,樊巧玲,王明艳等.左归丸对SD大鼠骨髓间充质干细胞增殖的影响[J].中国实验方剂学杂志,2011,17(13)：145-148
[63]Jiang XH, Yie SM, Zhen X, et al. Effect of You Gui Wan on mouse sperm fertilising ability in vivo and in vitro[J]. Andrologia.2013 Mar 6
[64]刘永琦,蔡玲,颜春鲁,等.补肾中药对大鼠BMSCs分泌NGF β、BDNF、VEGF功能的影响[J].免疫学杂志,2010,26(10)：850-853
[65]黄勇,黄秀深,胡一梅,等.“左归丸”配伍规律对骨髓源成体干细胞定向分化的调控作用[J].成都中医药大学学报,2010,33(1)：48-52
[66]胡黎平,涂平生,谢富康,等.补肾方剂对大鼠骨髓基质干细胞增殖与分化的影响[J].中国药物与临床,2007,7(5)：329-331
[67]陈东风,杜少辉,李伊为,等.龟板对局灶性脑缺血后神经干细胞的作用[J].广州中医药大学学报,2001,18(4)：328-331
[68]张进,徐志伟.补肾法诱导间充质干细胞向神经方向分化研究[J].现代医院,2004,4(9)：15-17
[69]曾建春,樊粤光,刘建仁,等.杜仲含药血清诱导骨髓间充质干细胞定向分化的实验研究[J].时珍国医国药,2009,20(9)：2136-2138
[70]张贤,蔡建平,张艳红.杜仲诱导大鼠间充质干细胞成骨分化中成骨与成脂相关转录因子的表达[J].中国组织工程研究与临床康复,2010,14(19)：3523-3526
[71]黄勇,吴建军,刘凯,等.熟地含药血清对骨髓间质干细胞向神经细胞分化的影响[JJ.中国老年学杂志,2011,31：3979-3981
[72]张永占,刘,占彦.补肾中药对女性生殖系统功能的影响[J].河南中医学院学报,2006,21(1)：85-88
[73]杨丽芸,杜惠兰,白静,等.补肾法、疏肝法对超促排卵小鼠卵母细胞数量及GDF-9表达的影响[J].中医杂志,2013,54(7)：597-604
[74]Karahuseyinoglu S, Cinar 0, Kilic E, et al. Biology of stem cells in human umbilical cord stroma:in situ and in vitro surveys[J]. Stem Cells,2007,25(2):319-331
[75]Hou Ke-dong, Xu Wen-jing, Zhang Li,et al.To study chondrogenic induction of mesenchymal stem cells derived from Wharton jelly of human umbilical cord[J]. Chinese Journal of Orthopaedics,2007,27 (12):930-935
[76]陈军,刘玉侠,姜文华,等.体外诱导人脐带间充质干细胞向肝样细胞的分化[J].中国组织工程研究与临床康复,2010,14(49)：9257-9261
[77]Can A,Karahuseyinoglu S. Concise review:human umbilical cord stroma with regard to the source of fetus-derived stem cells[J]. Stem Cells,2007,25(11).2886-2895
[78]Mitchell K E,Weiss M L.Mitchell B M, et al. Matrix cells from Whar-ton" s jelly form neurons and glia[J].Stem Cells,2003,21(1):50-60
[79]Dyce PW, Liu J, Tayade C, et al. In vitro and in vivo germ line potential of stem cells derived from newborn mouse skin[J]. PLoS One.2011,6(5):1-14
[80]Dyce PW, Wen L, Li J. In vitro germline potential of stem cells derived from fetal porcine skin[J]. Nature Cell Biology,2006,8(4):384-90
[81]Danner S,Kajahn J, Geismann C, et al.Derivation of oocyte-like cells from a clonal pancreatic stem cell line[J].Mol Hum Reprod,2007,13(1):11-20.
[82]Johnson J, Bagley J, Skaznik-Wikiel M, et al. Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood[J]. Cell,2005, 122(2):303-315
[83]常秀峰,杜惠兰,高星,等.补肾调经方对输卵管性不孕症患者体外受精-胚胎移植超排卵周期GDF-9的影响[J].中国中西医结合杂志,2011,31(6)：780-783
[84]饶研文,卢雪芳,罗明,等.71例克氏综合征患者血清性激素和游离睾酮检测分析[J].现代诊断与治疗,2009,20(3)：174
[85]张四林,杨菁,尹太郎,等.卵泡液中VEGF水平对卵母细胞成熟及早期胚胎发育的影响[J].武汉大学学报(医学版),2008,29(1)：60-62
[86]张雪青,张李雅,徐春泉,等.少精子症患者血清和精液中游离睾酮和双氢睾酮水平的 测定及临床意义[J].中国优生与遗传杂志,2007,15(2)：98-104
[87]牛小平,韩真,何池义,等.EGF、VEGF在常见消化道肿瘤患者血清中的表达及意义[J].现代肿瘤医学,2007,15(5)：668-669
[88]Balasch J,Guimera M, Pasarell OM, et al. Adrenomedullin and vascular endothelial growth factorproduction by follicular fluid macrophages and granulose cells [J]. Hum Reprod,2004,19(4):808-814
[89]Luo H, Kimura K, Aoki M,, et al. Effect of vascular endothelial growth factor on maturation, fertilization and developmental competence of bovine oocytes [J]. Veterinary Medical Science,2002,9:803-806
[90]Hailing Luo, Kimura K, M, Aoki, et al. Vascular Endothelial Growth Factor (VEGF) Promotes the Early Development of Bovine Embryo in the Presence of Cumulus Cells [J]. Veterinary Medical Science,2002,11:967-971
[91]董晓晨,王稳,罗永,等.血管内皮生长因子对猪卵母细胞体外成熟的影响研究[J].中国农学通报2012,28(23)：50-55
[92]夏蕾,张岭,吴坤.卵母细胞成熟过程MPF的调控和MPF的作用底物[J].疾病控制杂志,2007,11(6)：614-617
[93]A Picard, S Galas, G Peaucellier, et al. Newly assembled cyclin B-cdc2 kinase is required to suppress DNA replication between meiosis I and meiosis II in starfish oocytes[J].EMBO,1996,15(14):3590-3598.
[94]彭佑共,杨罗艳.Hela细胞MPF对未成熟卵母细胞的体外促成熟作用分析[J].医学临床研究,2008,25(9)：1624-1627
[95]沈自尹,黄建华.从淫羊藿激活内源性干细胞探讨“肾藏精”的科学涵义[J].中医杂志,2010,51(1)：8-10
[96]Yong-jun Wang, Ruth M Belf lower, Yu-Feng Dong, et al. Runxl/AML1/Cbf a 2 mediates onset of mesenchymal cell differentiation toward chondrogenesis[J]. Journal of Bone and Mineral Research,2005,20(9):1624-1636
[97]Do Y Soung, Yufeng Dong, Yong-Jun Wang, et al. Runx3/AML2/Cbf a 3 regulates early and late chondrocyte differentiation [J]. Journal of Bone and Mineral Research,2007, 22(8):1260-1270
[98]Yvonne A R White, Dori C Woods, Yasushi Takai, et al. Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women [J]. Nature Medicine,2012,18:413-421
[1]Hubner K, Fuhrmann G, Christenson LK, et al. Derivation of oocytes from mouse embryonic stem cells[J]. Science,2003,300(5623):1251-1256
[2]Lacham. Kaplan 0, ChyH, Trounson A. Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes[J]. Stem Cells,2005; 24(2):266-273
[3]Novak I, Lightfoot D A, Wang H, et al. Mouse embryonic stem cells form follicle-like ovarian structures but do not progress through meiosis[J], Stem Cells,2006,24: 1931-1936
[4]Clark AT, BodnarMS, FoxM, et al. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro [J].Hum MolGenet,2004,13:727-739
[5]Johnson J, Bagley J, Skaznik-Wikiel M, et al. Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood[J]. Cell,2005, 122(2):303-315
[6]Dyce PW, Wen L, Li J. In vitro germline potential of stem cells derived from fetal porcine skin[J]. Nature Cell Biology,2006,8(4):384-90
[7]Danner S,Kajahn J,Geismann C, et al. Derivation of oocyte-like cells from a clonal pancreatic stem cell line[J].Mol Hum Reprod,2007,13(1):11-20.
[8]Dyce PW, Liu J, Tayade C, et al. In vitro and in vivo germ line potential of stem cells derived from newborn mouse skin[J].PLoS One,2011,6(5):1-14
[9]杨文成,董有海.人脐带间充质干细胞的研究进展[J].组织工程与重建外科杂志,2010,6(5)：292-294
[10]孙国栋,李志忠.人脐带间充质干细胞在组织工程中的研究进展[J].现代生物医学进展,2009,9(12)：2389-2390
[11]Mareschi K, I Ferrero, D Rustichelli, et al. Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow[J]. Cell Biochem, 2006,97(4):744-754.
[12]Choumerianou DM, G Martimianaki, E Stiakaki, et al. Comparative study of stemness characteristics of mesenchymal cells from bone marrow of children and adults[J]. Cytotherapy,2010,12:881-887.
[13]李彩霞.人脐带基质干细胞体外向雌性生殖细胞分化的潜能[J].中国组织工程研究与临床康复,2010,14(40)：7583-7587
[14]吕品,何玲,柴惠霞.类胚体诱导脐带间充质干细胞向生殖细胞的分化[J].中国组织 工程研究,2012,16(23)：4217-4221
[15]Hong SH, Maghen L, Kenigsberg S, et al. Ontogeny of human umbilical cord peri vascular cells:molecular and fate potential changes during gestation. Stem Cells Dev.2013,5(14)