人脐带源间充质干细胞生物学特性及移植免疫学研究
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摘要
Friedenstein等发现的骨髓MSCs有着巨大生物学潜能及应用前景,而其高度污染性及随年龄增加而扩增与分化能力降低、创伤性操作等缺点限制了其广泛使用。2003年HUC-MSCs的成功提取及相关研究证实其具有骨髓MSCs的特点并具有来源充分、提取简单、增殖旺盛、不悖伦理、无创操作等优势。但HUC-MSCs成为骨组织工程学种子细胞面临是否存在同种异体移植排斥问题。全面研究其移植免疫学特性成为HUC-MSCs是否具有临床应用前景的关键。
本课题成功对HUC-MSCs进行提取,观察其生物学特征及体外成骨诱导,对诱导体系的变化进行动态观察,检测其不同条件下对同种异体白细胞抗原、B7因子的表达,设定多重因素下的MLR,对临床应用前后及MLR反应前后T细胞亚型及细胞因子变化进行分析,并对其免疫学机制进行探讨,为临床应用提供直接的理论依据。结论:HUC-MSCs为MSCs家族中一员并具有增殖旺盛、传代稳定、体外诱导成骨高效、同种异体移植的免疫逃逸及免疫调节能力,可能成为骨组织工程学理想的种子细胞。
Preface:
Bone defection that caused by complicated fracture and bone tumor is still a challenge to orthopedic surgery. The question that autogenous bone limited , undering traumatic manipulate and artificial bone expensive perplexed orthopedist and patients. To research ideal seed cells was carrying out after proposed conception of Bone Tissue Engineering. MSCs were thought to be have the huge biology potency and application prospect after MSCs were found. However high bacterial contamination, traumatic operation, amplification and differentiation potential degrade with age have becoming disadvantages without debate. MSCs that from others tissue also have different shortages and incompetence to become ideal bone tissue seed cells. In 2003, HUC-MSCs extract succeed and the research confirmed that it not only have characteristic of MSCs but have the advantages of sufficient resource, high proliferation, without ethics dispute, without traumatic manipulate as well. If HUC-MSCs will be applied in clinic in future it must face up to the question that allogeneil transplantation immunity. whether or not HUC-MSCs can be applying in clinic depends on systematic investigate immunology characteristic.
This topic divided into three parts: Firstly, through the research of extract cells from tissue lumps adherence to obtain cells and observing the morphous, contrasting the growth curve of different generations, surface antigen markers and dynamic observed changes of the cells morphous, quantity during induce ossify. The results of ossify were analysis through ALP dyeing, express of I type colloge, calcium tuberosis. At last, we confirmed that wether or not all of the results about the cells that we extracted possessed the characteristis of MSCs. Especially pay attention to the characteristis of HUC-MSCs about simply extraction, proliferation prosperity, go down to the future generation stabilization and ossify competent. and identified HUC-MSCs consistent with good seed cells characteristic of bone tissue engineering. Secondly and thirdly were the investigation of HUC-MSCs transplantation immunology characteristic. That divided two parts included vitro and vivo. 1) Vitro part: Through detected the HLA-ABC, HLA-DR, B7 factors under different conditions and MLR that was setting different state to investigate whether or not HUC-MSCs have the characteristic that can escape immunologic response and to possess immunoregulation potential. 2) Vivo part: Observed clinical reflection of 8 patients that transplanted HUC-MSCs in vivo by different dose. through ELISA, determined the change of cells factor before and after transplanted and contrasting with cytokine that within blood before MLR and supernatant after response. Applied flow cytometry to analysis leukomonocyte subset change before and after transplanted. synthesis analysis results of vivo and vitro. Investigated HUC-MSCs characteristic of escaping immunologic response and immunoregulation functions after transplanting to human, and analysis the mechanism of transplant. Concrete procedure as follow:
1. Extraction and bionomics of HUC-MSCs
1.1 Objective Extracted HUC-MSCs from umbilical core tissue and confirmed that HUC-MSCs posses MSCs characteristic through cultivate, passage, depicting the growth curve, expression of surface markers and induce ossify. HUC-MSCs also have the characteristic that easy to obtain, productive, quickly ossify, and so on.
1.2 methods Obtained HUC-MSCs through tissue lumps adherence culture, observeing the morphous characteristic, applying MTT to detect the proliferation of 2nd、5th、9th generations in different time and drawing growth curve to contrast difference of different generations; Detected surface signs characteristic by flow cytometer, Vitro ossify inducted under seting up control group; The result were measured by alkaline phosphatase staining, alizarin red staining, immunohistochemistry express of I type collogen.
1.3 Result We observed that cells appearance around tissue piece after 7d of tissue piece adherence. They present long fusiform and formation colony growth after 15d planted. according to 1:3 ratio go down to the future generation average 5d. Without change of morphous and proliferation state before 20th generation. The growth curve of 2nd、5th、9th generations HUC-MSCs roughly identify, presenting parabola shape: logarithmic growth after 2d~3d latent phase, 7d~8d present platform phase. HUC-MSCs positive for CD105 and CD44, negative for CD34 and CD45 by flow cytometry. At 7dth after induced, we can see that quantity of HUC-MSCs are decrease in induced groups contrasting with blanks and the cells morphous becoming shorten change from long fusiform to polygon. Multiplicity calcium noeud could be seen at the induced plate and without changing of morphous and HUC-MSCs present long fusiform and formation colony grow intensively at blanks. There are not calcium noeud appeared. On 14dth after induced, we can see that the quantity of cells progress decrease and morphous change from shorten fusiform to cube shape, plenty of pedes spurii stretch out, considerable calcium noeud was seen in induced groups; in blanks groups without morphous change and HUC-MSCs presented colony more intensively growth than 7dth, without calcium noeud was seen. There were red calcium noeud among cells after alizarin red stain and without calcium noeud besides compact arrange cells in blank groups. by AKP stain, we can see that cell nucleus present uniform blue , there were intracytoplasm positive bead with dark blue color located the side of cellular nucleu, without dark blue bead in blank groups. In I type collogen, immunohistochemistry Stain, we found that cell nucleus present uniform blue, there were intracytoplasm positive bead with dark brown color located the side of cellular nucleu.
1.4 Conclusion The morphous, growth characteristic, surface markets of the cells that we obtaind through tissue lumps adherence consistent with the characteristic of MSCs. The change that cells morphous, cells quantity and the results of immunohistochemistry through vitro induced ossify differentiate observed we can conclusion that HUC-MSCs have the characteristic of ossific vitro in short time and effective, those change did not taken place in blank groups without the change of morphous, growth characteristic, and so on. They consistent with character of bone tissue engineering excellent seed.
2. Immunology characteristic experiment About HUC-MSCs in vitro
2.1 Objective Analyze the expression of HUC-MSCs to allogenic HLA antigen and B7 factor, carrying out model experiment in vitro to verify if can arouse the proliferation to allogenic leukomonocyte,and possess immunological regulation effection.
2.2 methods Through flow cytometry to detect the expreesion of HLA-ABC, HLA-DR and CD80, CD86 about 5th generation HUC-MSCs that normal and pre-stimulate byγ-INF. Analysis the expression and change in order to judged allogeneil graft immunogenicity.Setting up two groups according to HUC- MSCs states above-mentioned carry out MLR experiment with health lymphomonocyte in vitro, two groups interfere with PHA and blank groups, detecting data detected by MTT after 7d cultivated,intragroup by analysis of variance and interclass by LSD to analyses the change in order to judge whether or not to cause proliferation of allogenic lymphocytic and possess capability about immunoregulation.
2.3 Result According to the results by flow cytometry we can see that HUC- MSCs positive express HLA-ABC, negtive express CD80, CD86, HLA-DR; HUC-MSCs that pre-stimulate byγ-INF increase the express of HLA-ABC, the expreesion of CD80、CD86、HLA-DR without marked change. MLR results to showed that: 5th generation HUC-MSCs which normal and pre-stimulate by γ-INF not only did not cause proliferation of allogenic lymphocytic but have the effection of inhibitory proliferation by PHA. Contrast beween interclass to show: the group that pre-stimulate byγ-INF have more effection on immunosuppr- essive action.
2.4 Conclusion HUC-MSCs have the character that immune escape and immunoregulation effection just like other MSCs,and notwithstanding stimulate byγ-INF, HUC-MSCs still escaping immune reaction. They maybe become ideal seed cells of bone tissue engineering be used allogeneil graft. they can also long-term present in body with flame mediator (γ-INF) and repeat used in sym-region, making more important effection.
3. Experiment in vivo and mechanism of immunoregulation to HUC-MSCs
3.1 Objective Through observation to 8 case patients about clinical appearance and various cytokine expreesion and leukomonocyte subtype ratio change after transplant investigated whether or not allogeneilt rejection really appear, in order to confirm whether or not HCU-MSCs have the effection of escape immune response and immunoregulatory function. Provide direct evidences for clinical application in the future. Contrasted cytokine change with MLR, analyses the results uniformity vivo and vitro,and analysis immunity mechanism of HCU-MSCs.
3.2 Methods
3.2.1 Obtained the blood plasma and cell supernatant from blood before MLR and product after reaction,applicated ELISA to detect dose change of IL-2, IL-10,γ-IFN.
3.2.2 Obtained venous blood of patients before and end after 7d HUC-MSCs transplanted, centrifuge and take blood plasma to detect the dose change of IL-2、γ-IFN、IL-10 by ELISA and contrast with the results of MLR reaction; Venous blood of two patients was detect to analyses the change of CD4+/CD8+ ratio.
3.3 Results: All of the results of 8 patients were evaluated by analysis of variance: we found that the contents of IL-2 without significant distinction, IL-10 increased andγ-IFN descend after MLR reaction, all of the results possess the statistical significance; two patients results of leukomonocyte subtype analyses by flow cytometry showed that CD4+/CD8+ ratios lightly descend after transplant, and still not caused CD4+/CD8+ to raised up on second time used after 7d of first transplant of HCU-MSCs.
3.4 Conclusion HUC-MSCs unable to evoke the immune response of host and have slight immunological regulation function after allogeneil graft, when transplant second time after transplant unable to evoke immunologic memory of host. The results possess uniformity through contrast the content of cytokine in vivo and vitro after transplant. The immune character of HUC-MSCs was relevant to content change of cytokine inbody.
Synthesized all of the research results, we can make conclusion that: HUC- MSC can be attract through tissue pieces adherence, proliferation prosperity, going down to the future generation stable, expressing superficies marks of MSCs, ossific quickly in vitro, consistent with the character of MSCs from others resource. HUC-MSC unable to evoke allogenic leukomonocyte amplifi- cation in vitro MLR, and have the effection of immunological regulation. The immune research in vivo confirmed that HUC-MSC possess character to escape immune response and effection of immunological regulation. The results in vitro coincide with in vivo, unable to evoke immune response of host when at second transplant.
本课题成功对HUC-MSCs进行提取,观察其生物学特征及体外成骨诱导,对诱导体系的变化进行动态观察,检测其不同条件下对同种异体白细胞抗原、B7因子的表达,设定多重因素下的MLR,对临床应用前后及MLR反应前后T细胞亚型及细胞因子变化进行分析,并对其免疫学机制进行探讨,为临床应用提供直接的理论依据。结论:HUC-MSCs为MSCs家族中一员并具有增殖旺盛、传代稳定、体外诱导成骨高效、同种异体移植的免疫逃逸及免疫调节能力,可能成为骨组织工程学理想的种子细胞。
Preface:
Bone defection that caused by complicated fracture and bone tumor is still a challenge to orthopedic surgery. The question that autogenous bone limited , undering traumatic manipulate and artificial bone expensive perplexed orthopedist and patients. To research ideal seed cells was carrying out after proposed conception of Bone Tissue Engineering. MSCs were thought to be have the huge biology potency and application prospect after MSCs were found. However high bacterial contamination, traumatic operation, amplification and differentiation potential degrade with age have becoming disadvantages without debate. MSCs that from others tissue also have different shortages and incompetence to become ideal bone tissue seed cells. In 2003, HUC-MSCs extract succeed and the research confirmed that it not only have characteristic of MSCs but have the advantages of sufficient resource, high proliferation, without ethics dispute, without traumatic manipulate as well. If HUC-MSCs will be applied in clinic in future it must face up to the question that allogeneil transplantation immunity. whether or not HUC-MSCs can be applying in clinic depends on systematic investigate immunology characteristic.
This topic divided into three parts: Firstly, through the research of extract cells from tissue lumps adherence to obtain cells and observing the morphous, contrasting the growth curve of different generations, surface antigen markers and dynamic observed changes of the cells morphous, quantity during induce ossify. The results of ossify were analysis through ALP dyeing, express of I type colloge, calcium tuberosis. At last, we confirmed that wether or not all of the results about the cells that we extracted possessed the characteristis of MSCs. Especially pay attention to the characteristis of HUC-MSCs about simply extraction, proliferation prosperity, go down to the future generation stabilization and ossify competent. and identified HUC-MSCs consistent with good seed cells characteristic of bone tissue engineering. Secondly and thirdly were the investigation of HUC-MSCs transplantation immunology characteristic. That divided two parts included vitro and vivo. 1) Vitro part: Through detected the HLA-ABC, HLA-DR, B7 factors under different conditions and MLR that was setting different state to investigate whether or not HUC-MSCs have the characteristic that can escape immunologic response and to possess immunoregulation potential. 2) Vivo part: Observed clinical reflection of 8 patients that transplanted HUC-MSCs in vivo by different dose. through ELISA, determined the change of cells factor before and after transplanted and contrasting with cytokine that within blood before MLR and supernatant after response. Applied flow cytometry to analysis leukomonocyte subset change before and after transplanted. synthesis analysis results of vivo and vitro. Investigated HUC-MSCs characteristic of escaping immunologic response and immunoregulation functions after transplanting to human, and analysis the mechanism of transplant. Concrete procedure as follow:
1. Extraction and bionomics of HUC-MSCs
1.1 Objective Extracted HUC-MSCs from umbilical core tissue and confirmed that HUC-MSCs posses MSCs characteristic through cultivate, passage, depicting the growth curve, expression of surface markers and induce ossify. HUC-MSCs also have the characteristic that easy to obtain, productive, quickly ossify, and so on.
1.2 methods Obtained HUC-MSCs through tissue lumps adherence culture, observeing the morphous characteristic, applying MTT to detect the proliferation of 2nd、5th、9th generations in different time and drawing growth curve to contrast difference of different generations; Detected surface signs characteristic by flow cytometer, Vitro ossify inducted under seting up control group; The result were measured by alkaline phosphatase staining, alizarin red staining, immunohistochemistry express of I type collogen.
1.3 Result We observed that cells appearance around tissue piece after 7d of tissue piece adherence. They present long fusiform and formation colony growth after 15d planted. according to 1:3 ratio go down to the future generation average 5d. Without change of morphous and proliferation state before 20th generation. The growth curve of 2nd、5th、9th generations HUC-MSCs roughly identify, presenting parabola shape: logarithmic growth after 2d~3d latent phase, 7d~8d present platform phase. HUC-MSCs positive for CD105 and CD44, negative for CD34 and CD45 by flow cytometry. At 7dth after induced, we can see that quantity of HUC-MSCs are decrease in induced groups contrasting with blanks and the cells morphous becoming shorten change from long fusiform to polygon. Multiplicity calcium noeud could be seen at the induced plate and without changing of morphous and HUC-MSCs present long fusiform and formation colony grow intensively at blanks. There are not calcium noeud appeared. On 14dth after induced, we can see that the quantity of cells progress decrease and morphous change from shorten fusiform to cube shape, plenty of pedes spurii stretch out, considerable calcium noeud was seen in induced groups; in blanks groups without morphous change and HUC-MSCs presented colony more intensively growth than 7dth, without calcium noeud was seen. There were red calcium noeud among cells after alizarin red stain and without calcium noeud besides compact arrange cells in blank groups. by AKP stain, we can see that cell nucleus present uniform blue , there were intracytoplasm positive bead with dark blue color located the side of cellular nucleu, without dark blue bead in blank groups. In I type collogen, immunohistochemistry Stain, we found that cell nucleus present uniform blue, there were intracytoplasm positive bead with dark brown color located the side of cellular nucleu.
1.4 Conclusion The morphous, growth characteristic, surface markets of the cells that we obtaind through tissue lumps adherence consistent with the characteristic of MSCs. The change that cells morphous, cells quantity and the results of immunohistochemistry through vitro induced ossify differentiate observed we can conclusion that HUC-MSCs have the characteristic of ossific vitro in short time and effective, those change did not taken place in blank groups without the change of morphous, growth characteristic, and so on. They consistent with character of bone tissue engineering excellent seed.
2. Immunology characteristic experiment About HUC-MSCs in vitro
2.1 Objective Analyze the expression of HUC-MSCs to allogenic HLA antigen and B7 factor, carrying out model experiment in vitro to verify if can arouse the proliferation to allogenic leukomonocyte,and possess immunological regulation effection.
2.2 methods Through flow cytometry to detect the expreesion of HLA-ABC, HLA-DR and CD80, CD86 about 5th generation HUC-MSCs that normal and pre-stimulate byγ-INF. Analysis the expression and change in order to judged allogeneil graft immunogenicity.Setting up two groups according to HUC- MSCs states above-mentioned carry out MLR experiment with health lymphomonocyte in vitro, two groups interfere with PHA and blank groups, detecting data detected by MTT after 7d cultivated,intragroup by analysis of variance and interclass by LSD to analyses the change in order to judge whether or not to cause proliferation of allogenic lymphocytic and possess capability about immunoregulation.
2.3 Result According to the results by flow cytometry we can see that HUC- MSCs positive express HLA-ABC, negtive express CD80, CD86, HLA-DR; HUC-MSCs that pre-stimulate byγ-INF increase the express of HLA-ABC, the expreesion of CD80、CD86、HLA-DR without marked change. MLR results to showed that: 5th generation HUC-MSCs which normal and pre-stimulate by γ-INF not only did not cause proliferation of allogenic lymphocytic but have the effection of inhibitory proliferation by PHA. Contrast beween interclass to show: the group that pre-stimulate byγ-INF have more effection on immunosuppr- essive action.
2.4 Conclusion HUC-MSCs have the character that immune escape and immunoregulation effection just like other MSCs,and notwithstanding stimulate byγ-INF, HUC-MSCs still escaping immune reaction. They maybe become ideal seed cells of bone tissue engineering be used allogeneil graft. they can also long-term present in body with flame mediator (γ-INF) and repeat used in sym-region, making more important effection.
3. Experiment in vivo and mechanism of immunoregulation to HUC-MSCs
3.1 Objective Through observation to 8 case patients about clinical appearance and various cytokine expreesion and leukomonocyte subtype ratio change after transplant investigated whether or not allogeneilt rejection really appear, in order to confirm whether or not HCU-MSCs have the effection of escape immune response and immunoregulatory function. Provide direct evidences for clinical application in the future. Contrasted cytokine change with MLR, analyses the results uniformity vivo and vitro,and analysis immunity mechanism of HCU-MSCs.
3.2 Methods
3.2.1 Obtained the blood plasma and cell supernatant from blood before MLR and product after reaction,applicated ELISA to detect dose change of IL-2, IL-10,γ-IFN.
3.2.2 Obtained venous blood of patients before and end after 7d HUC-MSCs transplanted, centrifuge and take blood plasma to detect the dose change of IL-2、γ-IFN、IL-10 by ELISA and contrast with the results of MLR reaction; Venous blood of two patients was detect to analyses the change of CD4+/CD8+ ratio.
3.3 Results: All of the results of 8 patients were evaluated by analysis of variance: we found that the contents of IL-2 without significant distinction, IL-10 increased andγ-IFN descend after MLR reaction, all of the results possess the statistical significance; two patients results of leukomonocyte subtype analyses by flow cytometry showed that CD4+/CD8+ ratios lightly descend after transplant, and still not caused CD4+/CD8+ to raised up on second time used after 7d of first transplant of HCU-MSCs.
3.4 Conclusion HUC-MSCs unable to evoke the immune response of host and have slight immunological regulation function after allogeneil graft, when transplant second time after transplant unable to evoke immunologic memory of host. The results possess uniformity through contrast the content of cytokine in vivo and vitro after transplant. The immune character of HUC-MSCs was relevant to content change of cytokine inbody.
Synthesized all of the research results, we can make conclusion that: HUC- MSC can be attract through tissue pieces adherence, proliferation prosperity, going down to the future generation stable, expressing superficies marks of MSCs, ossific quickly in vitro, consistent with the character of MSCs from others resource. HUC-MSC unable to evoke allogenic leukomonocyte amplifi- cation in vitro MLR, and have the effection of immunological regulation. The immune research in vivo confirmed that HUC-MSC possess character to escape immune response and effection of immunological regulation. The results in vitro coincide with in vivo, unable to evoke immune response of host when at second transplant.
引文
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