目的应用蛋白质组学实验方法对在异体异位生长发育的新生小鼠睾丸组织进行差异蛋白分析,从而对睾丸组织体外移植模型用于生精细胞发育研究的可行性提供进一步的理论依据。方法以新生小鼠睾丸组织为供体,免疫缺陷小鼠为受体进行组织移植...目的应用蛋白质组学实验方法对在异体异位生长发育的新生小鼠睾丸组织进行差异蛋白分析,从而对睾丸组织体外移植模型用于生精细胞发育研究的可行性提供进一步的理论依据。方法以新生小鼠睾丸组织为供体,免疫缺陷小鼠为受体进行组织移植,移植8周后收集移植物组织,采用双向凝胶电泳(2-DE)分离移植物组织总蛋白,同时分离8周正常小鼠睾丸组织总蛋白作对比分析,运用Im ageM aster 2D E lite 5.0图像分析软件识别移植物组织与正常组织差异表达的蛋白质点,应用基质辅助电离解析飞行时间质谱(MALD I-TOF-MS)获取肽质量指纹图谱(PMF),检索数据库鉴定差异表达的蛋白质点。结果正常睾丸组织和移植物组织双向电泳图谱的平均蛋白质点数分别为(1 326±15)个和(1 561±20)个,3次重复实验的蛋白质点位置重复性较好。通过比较两者的双向凝胶电泳图谱,得到表达差异量较大的蛋白质点21个。选取6个仅在正常睾丸组织中有表达的蛋白质点进行质谱和生物信息学分析,鉴定出的6种蛋白质,除血红蛋白α1链(Hbα-α1)外,其他5种蛋白均为在小鼠睾丸中有较高表达的蛋白质,包括A激酶锚定蛋白结合的精子蛋白(ASP)、磷脂过氧化氢物谷胱甘肽过氧化物酶(GPX4)、精子蛋白(Sp17)、天门冬酰胺酶样蛋白(ASRGL1)和过氧化物氧化还原因子4(Prdx4)。结论在本实验中,从异体异位生长发育成熟的新生小鼠睾丸组织中检测到5种在睾丸中特异性高表达蛋白质的缺失,这些蛋白质主要在精子运动、精子顶体形成以及精子的受精等功能中发挥作用。展开更多
Objective： To evaluate the dltterentlatlon ot human umbilical cord blood ceils into hepatocyte-like cells. Methods： Mononuclear cells （MNCs） derived from human umbilical cord blood were isolated using Ficoll. The ...Objective： To evaluate the dltterentlatlon ot human umbilical cord blood ceils into hepatocyte-like cells. Methods： Mononuclear cells （MNCs） derived from human umbilical cord blood were isolated using Ficoll. The experiment was derived into 3 categories： （1） MNCs co-cultured with 50 mg minced liver tissue separated by a trans-well membrane and then collected at 0 h, 24 h, 48 h and 72 h; （2） MNCs cultured along supplemented with 100 ml/L FBS, 100 μ/ml penicillin, 100 μg/ml streptomycin, 4.7 μg/ml linoleic acid, 1×ITS, 10^-4 mol/L L-Ascorbic acid 2-P and a combination of FGF4 （100 ng/ml） and HGF （20 ng/mL）. Cells were then collected at 0 d and 16 d to examine the expression profile of hepatocyte correlating markers; （3） 0.2-0.3 ml of MNCs with a cell density of 2×10^7/ml were transplanted into prepared recipient mice [n=12, injected with 0.4 ml/kg （20%） CCl4 and 150 ng/kg 5-fluorouracil （5-Fu） prior the transplant 24 h and 48 h, respectively] via injection through tail vein. Mice were sacrificed 4 weeks after transplantation. The hepatocyte correlating mRNAs and proteins were determined by RT-PCR, immunohistochemical analysis and immunoflurence technique. Results： （1） After 72 h, a number of glycogen positive stained cells were observed with MNCs co-cultured with damaged mouse liver tissues. The expression of hepatocyte markers, human albumin （ALB）, α-fetal protein （AFP） and human GATA4 mRNA and proteins were detected by RT-PCR and immunohistochemistry as well. For the confirmation, the DNA sequencing of PCR products was performed. In control groups, MNCs co-cuhured with normal mouse hepatocytes or MNCs cultured alone, all markers remained negative. （2） In growth factor supplemented culture system, MNCs developed into larger volume with richer cytoplasm and binucleation after 16 d. Positive expression of ALB, AFP, CK18 and CK19 mRNA were detected with RT-PCR, and ALB positive staining was observed by immunocytochemistry as well. In contrast, MNCs cultured without exogenous growth factors scarcely attached to the culture dish and ALB mRNA was not detected. （3） In transplantation experiment, both of ALB and AFP mRNA were detected by RT-PCR and HSA, PCNA and ALB positive staining were observed in the livers of recipient mice by immunocytochemistry. Conclusion： MNCs from human umbilical cord blood could convert into hepatocyte-like ceils in 3 different ways, indicating their potential use in the clinic applications for the treatment of human liver diseases.展开更多
文摘目的应用蛋白质组学实验方法对在异体异位生长发育的新生小鼠睾丸组织进行差异蛋白分析,从而对睾丸组织体外移植模型用于生精细胞发育研究的可行性提供进一步的理论依据。方法以新生小鼠睾丸组织为供体,免疫缺陷小鼠为受体进行组织移植,移植8周后收集移植物组织,采用双向凝胶电泳(2-DE)分离移植物组织总蛋白,同时分离8周正常小鼠睾丸组织总蛋白作对比分析,运用Im ageM aster 2D E lite 5.0图像分析软件识别移植物组织与正常组织差异表达的蛋白质点,应用基质辅助电离解析飞行时间质谱(MALD I-TOF-MS)获取肽质量指纹图谱(PMF),检索数据库鉴定差异表达的蛋白质点。结果正常睾丸组织和移植物组织双向电泳图谱的平均蛋白质点数分别为(1 326±15)个和(1 561±20)个,3次重复实验的蛋白质点位置重复性较好。通过比较两者的双向凝胶电泳图谱,得到表达差异量较大的蛋白质点21个。选取6个仅在正常睾丸组织中有表达的蛋白质点进行质谱和生物信息学分析,鉴定出的6种蛋白质,除血红蛋白α1链(Hbα-α1)外,其他5种蛋白均为在小鼠睾丸中有较高表达的蛋白质,包括A激酶锚定蛋白结合的精子蛋白(ASP)、磷脂过氧化氢物谷胱甘肽过氧化物酶(GPX4)、精子蛋白(Sp17)、天门冬酰胺酶样蛋白(ASRGL1)和过氧化物氧化还原因子4(Prdx4)。结论在本实验中,从异体异位生长发育成熟的新生小鼠睾丸组织中检测到5种在睾丸中特异性高表达蛋白质的缺失,这些蛋白质主要在精子运动、精子顶体形成以及精子的受精等功能中发挥作用。
基金Supported by the Shenzhen Science & Technology Planning Program (No. 200204109, No. JH200505270412B)
文摘Objective： To evaluate the dltterentlatlon ot human umbilical cord blood ceils into hepatocyte-like cells. Methods： Mononuclear cells （MNCs） derived from human umbilical cord blood were isolated using Ficoll. The experiment was derived into 3 categories： （1） MNCs co-cultured with 50 mg minced liver tissue separated by a trans-well membrane and then collected at 0 h, 24 h, 48 h and 72 h; （2） MNCs cultured along supplemented with 100 ml/L FBS, 100 μ/ml penicillin, 100 μg/ml streptomycin, 4.7 μg/ml linoleic acid, 1×ITS, 10^-4 mol/L L-Ascorbic acid 2-P and a combination of FGF4 （100 ng/ml） and HGF （20 ng/mL）. Cells were then collected at 0 d and 16 d to examine the expression profile of hepatocyte correlating markers; （3） 0.2-0.3 ml of MNCs with a cell density of 2×10^7/ml were transplanted into prepared recipient mice [n=12, injected with 0.4 ml/kg （20%） CCl4 and 150 ng/kg 5-fluorouracil （5-Fu） prior the transplant 24 h and 48 h, respectively] via injection through tail vein. Mice were sacrificed 4 weeks after transplantation. The hepatocyte correlating mRNAs and proteins were determined by RT-PCR, immunohistochemical analysis and immunoflurence technique. Results： （1） After 72 h, a number of glycogen positive stained cells were observed with MNCs co-cultured with damaged mouse liver tissues. The expression of hepatocyte markers, human albumin （ALB）, α-fetal protein （AFP） and human GATA4 mRNA and proteins were detected by RT-PCR and immunohistochemistry as well. For the confirmation, the DNA sequencing of PCR products was performed. In control groups, MNCs co-cuhured with normal mouse hepatocytes or MNCs cultured alone, all markers remained negative. （2） In growth factor supplemented culture system, MNCs developed into larger volume with richer cytoplasm and binucleation after 16 d. Positive expression of ALB, AFP, CK18 and CK19 mRNA were detected with RT-PCR, and ALB positive staining was observed by immunocytochemistry as well. In contrast, MNCs cultured without exogenous growth factors scarcely attached to the culture dish and ALB mRNA was not detected. （3） In transplantation experiment, both of ALB and AFP mRNA were detected by RT-PCR and HSA, PCNA and ALB positive staining were observed in the livers of recipient mice by immunocytochemistry. Conclusion： MNCs from human umbilical cord blood could convert into hepatocyte-like ceils in 3 different ways, indicating their potential use in the clinic applications for the treatment of human liver diseases.
