基于纤维蛋白胶支架材料的可注射组织工程化心肌的研究
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摘要
目的：本研究旨在以脂肪来源的干细胞(ADSCs)作为种子细胞来源,并以纤维蛋白胶可注射支架材料作为载体,开展用于可注射性心肌组织工程修复心肌梗死的研究。本研究包括：1)体外分离培养大鼠脂肪干细胞(ADSCs)并研究其基本生物学特性,探讨其为可注射的组织工程化心肌治疗心肌梗死提供种子细胞的可行性；2)探讨体外纤维蛋白胶中三维培养的大鼠脂肪干细胞(ADSCs)的增殖和存活性,并进行纤维蛋白胶体内组织形容性研究和生物降解性能评价,为可注射的组织工程化心肌治疗心肌梗死提供实验依据；3)以可注射纤维蛋白胶作为载体携带大鼠脂肪干细胞心梗部位移植,对其心梗损伤修复能力进行进一步的研究。
方法：1)本课题第一部分体外分离培养大鼠脂肪来源的干细胞并进行传代培养,流式细胞术鉴定CD90、CD29、CD34及CD45的表达及检测细胞周期；绘制ADSCs的生长曲线；通过细胞形态观察、特异性染色及免疫组化方法来检测脂肪干细胞向成骨、成脂肪细胞及心肌细胞诱导分化的能力；2)本课题第二部分采用不同浓度的凝血酶与纤维蛋白原进行配比,摸索最佳的浓度与胶凝固时间；体外将ADSCs与不同浓度的纤维蛋白胶共培养,摸索最适宜的纤维蛋白原浓度；用噻唑蓝(MTT)比色法及LIVE/DEAD荧光双染色检测ADSCs在纤维蛋白胶中的增殖和存活性；随机选取SD大鼠8只,将纤维蛋白胶注射于大鼠心尖部,不同时间点取材,通过组织学染色检测其组织相容性及体内生物降解性。3)课题第三部分以可注射性纤维蛋白胶为载体,脂肪干细胞为种子细胞,体外构建可注射性工程化心肌并进行大鼠心梗部位移植。90只SD大鼠建立心梗模型,存活动物随机分为4组：单纯注射PBS组(10只)、单纯注射Fibrin组(10只)、单纯注射ADSCs组(18只)、注射Fibrin+ADSCs组(18只)。模型建立后1周,于梗死边缘区移植4,6二乙酰基-2-苯基吲哚(DAPI)标记的100uL 5×106个ADSCs、单纯的纤维蛋白胶和PBS。Fibrin+ADSCs组用纤维蛋白胶加ADSCs注射。术后24小时,ADSCs组和Fibrin+ADSCs组各有8只动物处死取材,观察移植细胞24h滞留率；余下所有各组动物则在术后4w,应用血流动力学方法和心脏超声方法检查心功能,随后处死动物取心肌标本做冰冻切片,测量心肌梗死面积、左心室室壁厚度、用DAPI、肌节辅肌动蛋白免疫组化双染法确定4周细胞的存活,免疫荧光检测心肌肌钙蛋白T(cTnT)、八因子相关抗原(vWAg)、α-平滑肌肌动蛋白(SMA)抗体；八因子染色法检测微血管密度。
结果：1)结果表明大鼠ADSCs表型特征为CD90、CD29阳性,CD34、CD45阴性；ADSCs能连续传代达15代以上,且在第7代仍保持较强的增殖能力；流式细胞术显示各代次的ADSCs约80%左右处于细胞周期的G0-G1期；ADSCs体外具有向成骨、成脂肪细胞分化的潜能,但向心肌细胞诱导分化的能力差。2)纤维蛋白原浓度为10mg/ml及凝血酶浓度为50iu/mL时成胶时间适宜,细胞生长状态好；MTT结果显示,纤维蛋白胶上的ADSCs增殖活性高于对照组(P<0.05),LIVE/DEAD荧光双染色显示ADSCs在纤维蛋白胶中生长良好,24h和72h的细胞存活率均在90%以上,死亡率小于7%；体内生物学评价结果说明纤维蛋白胶具有良好的组织相容性及生物降解性。3)移植后24h,纤维蛋白胶联合ADSCs移植显著提高了移植细胞在注射区域的滞留率(P<0.01),移植后4周,Fibrin+ADSCs组心肌梗死区的存活细胞数量多于单纯细胞移植组(P<0.01)。Fibrin+ADSCs组心脏超声的各个指标及血流动力学显示的LVSP,+dp/dtmax均高于其他三组(P<0.01)。Fibrin+ADSCs组心肌梗死面积明显低于ADSCs组和Fibrin组(均P<0.01),室壁厚度大于ADSCs组和Fibrin组(均P<0.01)；Fibrin+ADSCs组胶原纤维的面积较其他组明显减少；免疫荧光结果表明脂肪干细胞移植大鼠心脏4周后,可以分化为cTnT阳性细胞、vWAg阳性细胞及SMA阳性细胞。Fibrin+ADSCs组梗死区的毛细血管密度显著高于ADSCs组、Fibrin组和对照组(均P<0.01)。
结论：1)大鼠脂肪干细胞体外易于分离培养、扩增迅速、增殖能力强,具有向成骨、成脂肪细胞诱导分化的潜能,第7代之前的细胞可以作为组织工程的种子细胞来源；2)大鼠脂肪干细胞在纤维蛋白胶中增殖和存活良好,纤维蛋白胶具有良好的细胞相容性、组织相容性及生物降解性,是心肌组织工程种子细胞较为理想的载体材料；3)以可注射纤维蛋白胶为载体携带脂肪干细胞心梗移植验证了纤维蛋白胶作为可注射性组织工程化心肌载体的可行性。这种基于可注射性支架材料的心肌组织工程研究对于缺血性心肌病的治疗具有潜在的临床应用价值。
Objective:The research is aimed at studying the usefulness of the combination of fibrin glue (as scaffold) and adiposederived stem cells (ADSCs) to treat myocardial infarction. The present study focuses on the following issues:1) To investigate a method of culture and isolation of rat adipose tissue-derived stem cells in vitro and explore their biological properties, and to discuss the feasibility of ADSCs as seed cells in injectable myocardial tissue engineering.2) To investigate the effects of the fibrin glue on the proliferation and viability of adipose tissue-derived stem cell of rat, and to evaluate the biocompatibility and biodegradability of the fibrin glue using histopathology staining in vivo, which provided the experiment evidence of injectable myocardial tissue engineering.3) Injectable cardiac tissue engineering aims to support cell-based therapies and enhance their efficacy for cardiac diseases. No research is devoted to studying the usefulness of the combination of fibrin glue (as scaffold) and adipose-derived stem cells (ADSCs) to treat myocardial infarction.
Methods:1) Firstly, the isolated ADSCs were cultured in vitro. The cell phenotype and generation cycle were detected by flow cytometry;growth curves of the cells were drawn with help of MTT assay;the characteristics of osteogenic and adipogenic and cardiomyocytes differentiation potential were investigated.2) Fibrin glue were prepared by combining fibrinogen with thrombin of different concentration for exploring suitable concentration and time. To Culture adipose tissue-derived stem cell and fibrin glue in vitro, the suitable concentration of fibrinogen was explored. The proliferation was tested by MTT assay and cell viability was assessed with LIVE/DEAD stain between ADSCs culture alone and ADSCs seeded fibrin glue. Fibrin glue was injected into the cardiac apex of 8 Rats, histopathology staining was performed to evaluate the biocompatibility and biodegradability of fibrin glue at different point in vivo.3) Fibrin glue was co-injected with ADSCs into the left ventricular wall of rat infarction models. Myocardial infarction was induced in 90 female rats using coronary artery ligation. Surviving rats that underwent ligation were randomly divided into 4 equal group, injections were performed along the border zone. 100μL of PBS was injected into the ischemic LV in control group (n=10).50μL of fibrinogen and 50μL of thrombin were simultaneously injected into ischemic myocardium in Fibrin group (n=10).5×106ADSCs in 100μL of PBS were injected into the infract in ADSCs group (n= 18,8 for 24-h cell retention observation).5×106ADSCs in 100uL of fibrin glue were injected into the infract in Fibrin+ADSCs group (n=18,8 for 24-h cell retention observation). Four weeks after the injection, the surviving rats underwent examination of heart functions by the echocardiography and hemodynamics. The rats were killed and their hearts were taken out to undergo immunohistochemistry with 4'6-diamidino-2-phenylindole (DAPI) and actin to measure the area of cardiac infarction. The heart infarcted size and wall thickness were calculated by masson trichrome staining. The differentiation of ADSCs in the infarcted heart was identified using cardiac troponin T (cTnT), vWAg antibody and smooth muscle a-actin (SMA). Blood vessel formation was detected by vWAg antibody.
Results:1) The ADSCs were positive for CD90 and CD29, but negative for CD34 and CD45;The ADSCs could be expanded for 15 passages, and showed active proliferation on generation 7 (P7), more than 80%cells of ADSCs were found at G0-G1 phase. The ADSCs were differentiate into osteogenic and adipogenic cells. However, ADSCs may not have the ability to differentiate into cardiomyocytes in vitro.2) The fibrin glue prepared by using l0mg/mL fibrinogen and 50iu/mL thrombin is suitable for the application in cardiac tissue engineering. ADSCs showed fast proliferation in fibrin glue group than the control group (P<0.05);ADSCs were successfully cultured in fibrin glue in vitro. The viability of ADSCs maintain>90%and the mortality of ADSCs maintain<7%at 24h and 72h. The biocompatibility and biodegradability of fibrin glue are good in the myocardium.3) After injection, both the.24h-cell retention and 4-week graft size were significantly higher and larger in the Fibrin+ADSCs group than those of the ADSCs group alone (P<0.1). The heart function improved significantly in the Fibrin+ ADSCs group compared with that of the ADSCs group 4 weeks after transplantation (P Conclusion:1) The Rat ADSCs was easy to isolate and culture in vitro. The proliferation ability of ADSCs was strong, especially before 7th passage. The ADSCs have the potential for osteogenic and adipogenic differentiation. The ADSCs would be a promising source of stem cells for tissue engineering.2) The proliferation and viability of ADSCs with fibrin glue were good. The biocompatibility and biodegradability of fibrin glue are good in the myocardium. Fibrin glue may work as a vehicle in deliverying ADSCs for injectable myocardial tissue engineering.3) The result from this study indicate that fibrin glue was potential injectable scaffolds that can be used to deliver stem cells to infarcted myocardium. The ADSCs with fibrin glue has the therapeutic potential to improve the function of infarcted hearts. The method of in situ injectable tissue engineering combining fibrin glue with ADSCs is promising clinically.
方法：1)本课题第一部分体外分离培养大鼠脂肪来源的干细胞并进行传代培养,流式细胞术鉴定CD90、CD29、CD34及CD45的表达及检测细胞周期；绘制ADSCs的生长曲线；通过细胞形态观察、特异性染色及免疫组化方法来检测脂肪干细胞向成骨、成脂肪细胞及心肌细胞诱导分化的能力；2)本课题第二部分采用不同浓度的凝血酶与纤维蛋白原进行配比,摸索最佳的浓度与胶凝固时间；体外将ADSCs与不同浓度的纤维蛋白胶共培养,摸索最适宜的纤维蛋白原浓度；用噻唑蓝(MTT)比色法及LIVE/DEAD荧光双染色检测ADSCs在纤维蛋白胶中的增殖和存活性；随机选取SD大鼠8只,将纤维蛋白胶注射于大鼠心尖部,不同时间点取材,通过组织学染色检测其组织相容性及体内生物降解性。3)课题第三部分以可注射性纤维蛋白胶为载体,脂肪干细胞为种子细胞,体外构建可注射性工程化心肌并进行大鼠心梗部位移植。90只SD大鼠建立心梗模型,存活动物随机分为4组：单纯注射PBS组(10只)、单纯注射Fibrin组(10只)、单纯注射ADSCs组(18只)、注射Fibrin+ADSCs组(18只)。模型建立后1周,于梗死边缘区移植4,6二乙酰基-2-苯基吲哚(DAPI)标记的100uL 5×106个ADSCs、单纯的纤维蛋白胶和PBS。Fibrin+ADSCs组用纤维蛋白胶加ADSCs注射。术后24小时,ADSCs组和Fibrin+ADSCs组各有8只动物处死取材,观察移植细胞24h滞留率；余下所有各组动物则在术后4w,应用血流动力学方法和心脏超声方法检查心功能,随后处死动物取心肌标本做冰冻切片,测量心肌梗死面积、左心室室壁厚度、用DAPI、肌节辅肌动蛋白免疫组化双染法确定4周细胞的存活,免疫荧光检测心肌肌钙蛋白T(cTnT)、八因子相关抗原(vWAg)、α-平滑肌肌动蛋白(SMA)抗体；八因子染色法检测微血管密度。
结果：1)结果表明大鼠ADSCs表型特征为CD90、CD29阳性,CD34、CD45阴性；ADSCs能连续传代达15代以上,且在第7代仍保持较强的增殖能力；流式细胞术显示各代次的ADSCs约80%左右处于细胞周期的G0-G1期；ADSCs体外具有向成骨、成脂肪细胞分化的潜能,但向心肌细胞诱导分化的能力差。2)纤维蛋白原浓度为10mg/ml及凝血酶浓度为50iu/mL时成胶时间适宜,细胞生长状态好；MTT结果显示,纤维蛋白胶上的ADSCs增殖活性高于对照组(P<0.05),LIVE/DEAD荧光双染色显示ADSCs在纤维蛋白胶中生长良好,24h和72h的细胞存活率均在90%以上,死亡率小于7%；体内生物学评价结果说明纤维蛋白胶具有良好的组织相容性及生物降解性。3)移植后24h,纤维蛋白胶联合ADSCs移植显著提高了移植细胞在注射区域的滞留率(P<0.01),移植后4周,Fibrin+ADSCs组心肌梗死区的存活细胞数量多于单纯细胞移植组(P<0.01)。Fibrin+ADSCs组心脏超声的各个指标及血流动力学显示的LVSP,+dp/dtmax均高于其他三组(P<0.01)。Fibrin+ADSCs组心肌梗死面积明显低于ADSCs组和Fibrin组(均P<0.01),室壁厚度大于ADSCs组和Fibrin组(均P<0.01)；Fibrin+ADSCs组胶原纤维的面积较其他组明显减少；免疫荧光结果表明脂肪干细胞移植大鼠心脏4周后,可以分化为cTnT阳性细胞、vWAg阳性细胞及SMA阳性细胞。Fibrin+ADSCs组梗死区的毛细血管密度显著高于ADSCs组、Fibrin组和对照组(均P<0.01)。
结论：1)大鼠脂肪干细胞体外易于分离培养、扩增迅速、增殖能力强,具有向成骨、成脂肪细胞诱导分化的潜能,第7代之前的细胞可以作为组织工程的种子细胞来源；2)大鼠脂肪干细胞在纤维蛋白胶中增殖和存活良好,纤维蛋白胶具有良好的细胞相容性、组织相容性及生物降解性,是心肌组织工程种子细胞较为理想的载体材料；3)以可注射纤维蛋白胶为载体携带脂肪干细胞心梗移植验证了纤维蛋白胶作为可注射性组织工程化心肌载体的可行性。这种基于可注射性支架材料的心肌组织工程研究对于缺血性心肌病的治疗具有潜在的临床应用价值。
Objective:The research is aimed at studying the usefulness of the combination of fibrin glue (as scaffold) and adiposederived stem cells (ADSCs) to treat myocardial infarction. The present study focuses on the following issues:1) To investigate a method of culture and isolation of rat adipose tissue-derived stem cells in vitro and explore their biological properties, and to discuss the feasibility of ADSCs as seed cells in injectable myocardial tissue engineering.2) To investigate the effects of the fibrin glue on the proliferation and viability of adipose tissue-derived stem cell of rat, and to evaluate the biocompatibility and biodegradability of the fibrin glue using histopathology staining in vivo, which provided the experiment evidence of injectable myocardial tissue engineering.3) Injectable cardiac tissue engineering aims to support cell-based therapies and enhance their efficacy for cardiac diseases. No research is devoted to studying the usefulness of the combination of fibrin glue (as scaffold) and adipose-derived stem cells (ADSCs) to treat myocardial infarction.
Methods:1) Firstly, the isolated ADSCs were cultured in vitro. The cell phenotype and generation cycle were detected by flow cytometry;growth curves of the cells were drawn with help of MTT assay;the characteristics of osteogenic and adipogenic and cardiomyocytes differentiation potential were investigated.2) Fibrin glue were prepared by combining fibrinogen with thrombin of different concentration for exploring suitable concentration and time. To Culture adipose tissue-derived stem cell and fibrin glue in vitro, the suitable concentration of fibrinogen was explored. The proliferation was tested by MTT assay and cell viability was assessed with LIVE/DEAD stain between ADSCs culture alone and ADSCs seeded fibrin glue. Fibrin glue was injected into the cardiac apex of 8 Rats, histopathology staining was performed to evaluate the biocompatibility and biodegradability of fibrin glue at different point in vivo.3) Fibrin glue was co-injected with ADSCs into the left ventricular wall of rat infarction models. Myocardial infarction was induced in 90 female rats using coronary artery ligation. Surviving rats that underwent ligation were randomly divided into 4 equal group, injections were performed along the border zone. 100μL of PBS was injected into the ischemic LV in control group (n=10).50μL of fibrinogen and 50μL of thrombin were simultaneously injected into ischemic myocardium in Fibrin group (n=10).5×106ADSCs in 100μL of PBS were injected into the infract in ADSCs group (n= 18,8 for 24-h cell retention observation).5×106ADSCs in 100uL of fibrin glue were injected into the infract in Fibrin+ADSCs group (n=18,8 for 24-h cell retention observation). Four weeks after the injection, the surviving rats underwent examination of heart functions by the echocardiography and hemodynamics. The rats were killed and their hearts were taken out to undergo immunohistochemistry with 4'6-diamidino-2-phenylindole (DAPI) and actin to measure the area of cardiac infarction. The heart infarcted size and wall thickness were calculated by masson trichrome staining. The differentiation of ADSCs in the infarcted heart was identified using cardiac troponin T (cTnT), vWAg antibody and smooth muscle a-actin (SMA). Blood vessel formation was detected by vWAg antibody.
Results:1) The ADSCs were positive for CD90 and CD29, but negative for CD34 and CD45;The ADSCs could be expanded for 15 passages, and showed active proliferation on generation 7 (P7), more than 80%cells of ADSCs were found at G0-G1 phase. The ADSCs were differentiate into osteogenic and adipogenic cells. However, ADSCs may not have the ability to differentiate into cardiomyocytes in vitro.2) The fibrin glue prepared by using l0mg/mL fibrinogen and 50iu/mL thrombin is suitable for the application in cardiac tissue engineering. ADSCs showed fast proliferation in fibrin glue group than the control group (P<0.05);ADSCs were successfully cultured in fibrin glue in vitro. The viability of ADSCs maintain>90%and the mortality of ADSCs maintain<7%at 24h and 72h. The biocompatibility and biodegradability of fibrin glue are good in the myocardium.3) After injection, both the.24h-cell retention and 4-week graft size were significantly higher and larger in the Fibrin+ADSCs group than those of the ADSCs group alone (P<0.1). The heart function improved significantly in the Fibrin+ ADSCs group compared with that of the ADSCs group 4 weeks after transplantation (P
引文
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