功能性自组装双亲多肽水凝胶支架联合脂肪间充质干细胞智能修兔骨性关节炎的实验研究

发布时间：2018-04-28 18:47

本文选题：双亲多肽 + 水凝胶　；参考：《华中科技大学》2014年博士论文

【摘要】：第一部分新型自组装双亲多肽水凝胶的合成目的：设计和合成促进细胞增殖和TGF-β粘附的自组装双亲多肽水凝胶,并检测其自组装形成凝胶后的超微结构。方法：合成的PRG和TB多肽粉剂分别溶解于去离子水中,终浓度为1%。然后再分别稀释成0.05%(m/v),分别取3ul的PRG,TB及两者等体积混合的多肽溶液。原子力显微镜检测多肽的超微结构。结果：成功合成PRG和TB双亲多肽分子,双亲多肽溶液PRG和TB混合自组装形成凝胶。原子力显微镜扫描提示：PRG/TB混合水凝胶的纳米纤维比PRG和TB要大,PRG,TB,PRG/TB纳米纤维的平均直径分别为(16.7±4.7nm),(18.0±1.3nm)和(28.3±5.6nm)。结论：双亲多肽PRG和TB可以混合自组装形成纳米级凝胶支架(PRG/TB),可以作为软骨组织工程的生物支架材料。第一1部分新骨性关节炎模型的研究目的：探讨通过注射MIA的方法建立一种能模拟自然病程引起的早,中期骨性关节炎模型,用以评价组织工程修复关节软骨的研究。方法：40只新西兰雄性大白兔子被随机分为三组。麻醉后,通过关节穿刺的方法,每组膝关节右侧注射100ul不同浓度的MIA(1mg/ml、3mg/ml和6mg/ml),左侧膝关节注射PBS作为对照组。分别于2周,4周和6周拍膝关节X线,并用墨汁染色比较大体形态,组织病理染色观察软骨组织的退变。结果：兔膝关节1mg/ml MIA注射组关节退变不明显,兔膝关节3mg/ml和6mg/ml MIA组病理染色评分和对照组比有统计学意义(P0.05)。但6mg/ml MIA注射组关节退变严重。结论：通过注射3mg/ml MIA的兔膝关节可以在六周内达到类似轻,中度骨性关节炎的关节退变,为评价组织工程修复软骨退变提供一个新的骨性关节炎动物模型参考。第三部分慢病毒载体融合蛋白TGF-β3和多肽水凝胶对脂肪间充质干细胞的生物学影响目的：构建出具有靶向治疗功能的新型TGF-β3融合蛋白LAP-MMP-mTGF-β3,并通过慢病毒载体包装后转染脂肪间充质干细胞(ADSCs),两者培养在多肽水凝胶支架内,以验证其可行性与靶向特异性。方法：消化法分离得到兔ADSCs,通过流式法鉴定ADSCs表面抗原及特殊染色法鉴定ADSCs多向分化能力。通过基因重组方法将LAP, mTGF-p3和基质金属蛋白酶(MMP)的酶切位点PLGLWA分别插入到真核质粒表达载体GV287,得到重组TGF-P3融合蛋白LAP-MMP-mTGF-β3的质粒。用慢病毒载体包装质粒后转染ADSCs,并培养在PRG/TB多肽水凝胶内。CCK8法检测增殖细胞增殖,钙黄绿素/碘化丙啶法检测细胞在凝胶内部的存活,Elisa法检测MMP酶刺激后上清液中的mTGF-p3。结果：成功分离培养出ADSCs,并且ADSCs培养在PRG/TB混合多肽水凝胶内可以提高增殖。慢病毒载体及PRG/TB多肽水凝胶对ADSCs的毒副作用小,并且MMP酶存在时可激活重组TGF-3融合蛋白释放活性的TGF-β3。结论：成功构建出慢病毒载体包装的新型融合蛋白LAP-MMP-mTGF-β3,慢病毒载体包装的新型融合蛋白转染的ADSCs与PRG/TB多肽水凝胶一起具有靶向性修复软骨缺损的应用前景。第四部分LV-mTGF-p3转染的脂肪间充质干细胞在双亲多肽水凝胶内诱导成软骨细胞的研究目的：探讨功能性自组装PRG/TB水凝胶支架与慢病毒包装的融合蛋白LAP-MMP-mTGF-β3转染的兔脂肪间充质干细胞(ADSCs)诱导成软骨的影响。方法：将慢病毒包装的融合蛋白LAP-MMP-mTGF-β3转染第三代ADSCs后,与功能性PRG/TB水凝胶支架共同培养在软骨细胞诱导液中,并增加10ng/m)基质金属蛋白酶-1(MMP-1)促进融合蛋白LAP-MMP-mTGF-p3释放TGF-β3,促进ADSCs分化成软骨细胞。3天后用Western blot检测TGF-P3在ADSCs中的表达。ADSCs诱导分化成软骨细胞21天后,分别用Real-time PCR和Western blot检测软骨细胞特异性基因Aggrecan (ACAN),II型胶原(COL2A1)和SOX-9的mRNA和蛋白的表达。结果：慢病毒包装融合蛋白LAP-MMP-mTGF-β3转染ADSCs效率能达到90%以上。3天后转染慢病毒的ADSCs成功检测到TGF-β3的表达。与未转染融合蛋白LAP-MMP-mTGF-β3的ADSCs相比,转染组明显促进了ADSCs向软骨细胞分化。21天后转染组与未转染组相比,ACAN、COL2A1和SOX-9mRNA表达分别增加2.7,2.4和1.0倍,蛋白表达分别增加2.4,1.57和0.89倍。结论：成功构建的慢病毒包装融合蛋白LAP-MMP-mTGF-p3转染ADSCs可促进ADSCs分化成软骨细胞,具有很好的修复软骨缺损的应用前景。第五部分功能性自组装多肽水凝胶联合LV-mTGF-β3转染的ADSCs修复骨性关节炎的研究目的：进一步验证PRG/TB自组装多肽水凝胶支架联合LV-mTGF-β3转染的兔脂肪间充质干细胞(ADSCs)在体内修复软骨的能力。方法：将慢病毒包装的融合蛋白LAP-MMP-mTGF-β3转染ADSCs后,与功能性PRG/TB自组装多肽水凝胶支架共同培养在软骨细胞诱导液中。21天后,在裸鼠背部分别种植不同组的ADSCs和多肽PRG/TB自组水凝胶混合物,观察多肽凝胶的毒副作用,4W后注射部位的组织用阿利新蓝染色和免疫组化染色检测成软骨的能力。新西兰大白兔用注射MIA方法完成骨性关节炎造模,然后每七天注射一次多肽水凝胶和慢病毒转染后的ADSCs混合物到骨性关节炎模型的膝关节,注射PBS作为试验对照组。8W后,组织染色观察多肽水凝胶联合重组TGF-p3蛋白转染的ADSCs修复关节的情况。结果：慢病毒包装的融合蛋白LAP-MMP-mTGF-β3转染ADSCs组在裸鼠皮下生成的软骨组织外形要大于其他组,阿利新蓝染色和免疫组化染色阳性表达明显高于其余组。注射多肽水凝胶和慢病毒转染后的ADSCs混合物组的病理染色结果显示明显改善了兔膝关节OA中软骨组织的退化。结论：功能性自组装多肽水凝胶联合慢病毒包装的重组TGF-β3蛋白转染的ADSCs能够在体内生成软骨组织,并明显改善OA中软骨组织退化的情况,具有很好的修复软骨缺损的应用前景。
[Abstract]:The first part is the synthesis of new self-assembled amphiphilic peptide hydrogels.
Objective: to design and synthesize self-assembly amphiphilic peptide hydrogels to promote cell proliferation and TGF- beta adhesion, and to detect the ultrastructure of self assembled gel.
Methods: the synthesized PRG and TB polypeptide powders were dissolved in deionized water respectively, the final concentration was 1%. and then diluted into 0.05% (m/v) respectively. The 3ul PRG, TB and the mixed polypeptide solutions were obtained respectively. The ultrastructure of the polypeptide was detected by atomic force microscopy.
Results: PRG and TB parent polypeptide molecules were successfully synthesized. The parent polypeptide solution PRG and TB were mixed to form the gel. The atomic force microscope scan suggested that the nanofibers of PRG/TB mixed hydrogel were larger than PRG and TB, the average diameter of PRG, TB and PRG/TB nanofibers was (16.7 + 4.7nm), (18 + 1.3nm) and (28.3 +).
Conclusion: the amphiphilic peptide PRG and TB can be self assembled to form nanoscale gel scaffolds (PRG/TB). They can be used as biomaterials for cartilage tissue engineering.
Study on the model of new osteoarthritis in the first 1 parts
Objective: to establish an early and middle term osteoarthritis model which can simulate the course of natural disease by injection of MIA to evaluate the repair of articular cartilage in tissue engineering.
Methods: 40 New Zealand male white rabbits were randomly divided into three groups. After anaesthesia, the right side of the knee joint was injected 100ul with different concentrations of MIA (1mg/ml, 3mg/ml and 6mg/ml) on the right side of the knee joint, and the left knee joint was injected with PBS as the control group. The knee joint X-ray was taken for 2 weeks, 4 and 6 weeks respectively, and the general morphology was compared with the ink stain. Pathological staining was used to observe the degeneration of cartilage tissue.
Results: the joint degeneration was not obvious in the 1mg/ml MIA injection group of the rabbit knee joint. The pathological staining score of the rabbit knee joint 3mg/ml and the 6mg/ml MIA group was statistically significant (P0.05), but the joint degeneration was serious in the 6mg/ml MIA injection group.
Conclusion: the rabbit knee joint by injection of 3mg/ml MIA can reach a joint degeneration similar to light and moderate osteoarthritis within six weeks, which provides a new animal model for osteoarthritis for tissue engineering repair of cartilage degeneration.
The third part is the biological effects of lentiviral vector fusion protein TGF- beta 3 and polypeptide hydrogel on adipose tissue derived mesenchymal stem cells.
Objective: to construct a novel TGF- beta 3 fusion protein LAP-MMP-mTGF- beta 3, which has the function of targeting therapy, and transfect fat mesenchymal stem cells (ADSCs) through the lentivirus vector, and the two are cultured in the peptide hydrogel scaffold to verify its feasibility and targeting specificity.
Methods: rabbit ADSCs was obtained by digestion method. ADSCs surface antigen was identified by flow method and special staining method was used to identify the multidirectional differentiation ability of ADSCs. The recombinant LAP, mTGF-p3 and matrix metalloproteinase (MMP) PLGLWA were inserted into the eukaryotic plasmid expression vector GV287 by gene recombination method, and the recombinant TGF-P3 fusion protein LAP-MMP-mT was obtained. Plasmids of GF- beta 3 were transfected with ADSCs by using lentivirus vector and cultured in PRG/TB polypeptide hydrogel to detect proliferation of proliferating cells. The survival of cells in the gel was detected by Calvin / propidium iodide method and mTGF-p3. in the supernatant after MMP stimulation was detected by Elisa method.
Results: ADSCs was successfully isolated and cultured, and ADSCs culture could increase the proliferation in PRG/TB mixed polypeptide hydrogel. The toxic and side effects of the lentivirus vector and the PRG/TB polypeptide hydrogel were small, and the MMP enzyme could activate the TGF- beta 3. of the recombinant TGF-3 fusion protein release activity.
Conclusion: a new fusion protein LAP-MMP-mTGF- beta 3 packed with lentivirus vector was successfully constructed. The new fusion protein transfected by lentivirus carrier ADSCs and PRG/TB polypeptide hydrogel have the prospect of targeted repair of cartilage defect with the PRG/TB polypeptide hydrogel.
The fourth part is the study of chondrocytes induced by LV-mTGF-p3 transfected adipose tissue derived mesenchymal stem cells in amphiphilic peptide hydrogels.
Objective: To investigate the effect of functional self-assembled PRG/TB hydrogel scaffold and lentivirus packaged fusion protein LAP-MMP-mTGF- beta 3 transfected on rabbit adipose mesenchymal stem cells (ADSCs) induced chondrogenic cartilage.
Methods: after transfecting the fusion protein LAP-MMP-mTGF- beta 3 of the lentivirus into third generation ADSCs, it was co cultured with the functional PRG/TB hydrogel scaffold in the chondrocyte inducer, and increased 10ng/m) matrix metalloproteinase -1 (MMP-1) promoting the release of TGF- beta 3 from the fusion protein LAP-MMP-mTGF-p3 and promoting ADSCs to differentiate into chondrocytes for.3 days with Western. The expression of TGF-P3 in ADSCs was detected by blot and.ADSCs was induced to differentiate into chondrocytes for 21 days. Real-time PCR and Western blot were used to detect the chondrocyte specific gene Aggrecan (ACAN), II collagen (COL2A1) and the expression of protein.
Results: the transfection of the lentivirus package fusion protein LAP-MMP-mTGF- beta 3 transfected to ADSCs could achieve the expression of TGF- beta 3 in the ADSCs transfected with lentivirus after more than 90% days.3. Compared with the ADSCs of the untransfected fusion protein LAP-MMP-mTGF- beta 3, the transfected group obviously promoted the ADSCs to cartilage cells to be transfected to the non transfected group, ACAN, compared with those in the untransfected group, ACAN. The expression of COL2A1 and SOX-9mRNA increased by 2.7,2.4 and 1 times respectively, and the protein expression increased by 2.4,1.57 and 0.89 times respectively.
Conclusion: the successfully constructed lentivirus package fusion protein LAP-MMP-mTGF-p3 transfected with ADSCs can promote the differentiation of ADSCs into chondrocytes, which has a good prospect of repairing cartilage defects.
The fifth part is functional self-assembled peptide hydrogel combined with LV-mTGF- beta 3 transfected ADSCs to repair osteoarthritis.
Objective: to further verify the ability of PRG/TB self assembled polypeptide hydrogel scaffold combined with LV-mTGF- beta 3 transfected rabbit adipose mesenchymal stem cells (ADSCs) to repair cartilage in vivo.
Methods: after transfecting the fusion protein LAP-MMP-mTGF- beta 3 of the lentivirus into ADSCs, the PRG/TB self assembled polypeptide hydrogel scaffold was cocultured with the functional PRG/TB self assembled polypeptide hydrogel scaffold for.21 days. The ADSCs and the polypeptide PRG/TB self group hydrogel mixture were planted in the back of the nude mice respectively, and the toxic and side effects of the polypeptide gel were observed and the 4W was injected after 4W injection. The tissue of the site was used to detect the chondrogenic ability with alino blue staining and immunohistochemical staining. New Zealand white rabbits were injected with MIA method to build the osteoarthritis model. Then a polypeptide hydrogel was injected once every seven days and the ADSCs mixture of lentivirus transfected to the knee joint of the osteoarthritis model, and PBS was injected into the control group after.8W. Tissue staining was used to observe the joint repair of collagen hydrogel combined with recombinant TGF-p3 protein transfected ADSCs.
Results: the expression of cartilage tissue in the subcutaneous tissue of group ADSCs transfected by lentivirus packed protein LAP-MMP-mTGF- beta 3 was greater than that of the other groups. The positive expression of alanine blue staining and immunohistochemical staining was significantly higher than that of the other groups. The pathological staining results of the ADSCs mixture group of the injected polypeptide hydrogel and lentivirus transfected showed obvious results. The degeneration of cartilage tissue in OA of rabbit's knee joint was improved.
Conclusion: the functional self-assembled polypeptide hydrogel combined with the recombinant TGF- beta 3 protein transfected with lentivirus can produce cartilage tissue in the body and obviously improve the degeneration of cartilage tissue in OA, which has a good prospect of repairing cartilage defect.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R684

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