本文選題：雙親多肽 + 水凝膠　； 參考：《華中科技大學(xué)》2014年博士論文

【摘要】：第一部分新型自組裝雙親多肽水凝膠的合成 目的：設(shè)計(jì)和合成促進(jìn)細(xì)胞增殖和TGF-β粘附的自組裝雙親多肽水凝膠,并檢測其自組裝形成凝膠后的超微結(jié)構(gòu)。 方法：合成的PRG和TB多肽粉劑分別溶解于去離子水中,終濃度為1%。然后再分別稀釋成0.05%(m/v),分別取3ul的PRG,TB及兩者等體積混合的多肽溶液。原子力顯微鏡檢測多肽的超微結(jié)構(gòu)。 結(jié)果：成功合成PRG和TB雙親多肽分子,雙親多肽溶液PRG和TB混合自組裝形成凝膠。原子力顯微鏡掃描提示：PRG/TB混合水凝膠的納米纖維比PRG和TB要大,PRG,TB,PRG/TB納米纖維的平均直徑分別為(16.7±4.7nm),(18.0±1.3nm)和(28.3±5.6nm)。 結(jié)論：雙親多肽PRG和TB可以混合自組裝形成納米級(jí)凝膠支架(PRG/TB),可以作為軟骨組織工程的生物支架材料。 第一1部分新骨性關(guān)節(jié)炎模型的研究 目的：探討通過注射MIA的方法建立一種能模擬自然病程引起的早,中期骨性關(guān)節(jié)炎模型,用以評(píng)價(jià)組織工程修復(fù)關(guān)節(jié)軟骨的研究。 方法：40只新西蘭雄性大白兔子被隨機(jī)分為三組。麻醉后,通過關(guān)節(jié)穿刺的方法,每組膝關(guān)節(jié)右側(cè)注射100ul不同濃度的MIA(1mg/ml、3mg/ml和6mg/ml),左側(cè)膝關(guān)節(jié)注射PBS作為對(duì)照組。分別于2周,4周和6周拍膝關(guān)節(jié)X線,并用墨汁染色比較大體形態(tài),組織病理染色觀察軟骨組織的退變。 結(jié)果：兔膝關(guān)節(jié)1mg/ml MIA注射組關(guān)節(jié)退變不明顯,兔膝關(guān)節(jié)3mg/ml和6mg/ml MIA組病理染色評(píng)分和對(duì)照組比有統(tǒng)計(jì)學(xué)意義(P0.05)。但6mg/ml MIA注射組關(guān)節(jié)退變嚴(yán)重。 結(jié)論：通過注射3mg/ml MIA的兔膝關(guān)節(jié)可以在六周內(nèi)達(dá)到類似輕,中度骨性關(guān)節(jié)炎的關(guān)節(jié)退變,為評(píng)價(jià)組織工程修復(fù)軟骨退變提供一個(gè)新的骨性關(guān)節(jié)炎動(dòng)物模型參考。 第三部分慢病毒載體融合蛋白TGF-β3和多肽水凝膠對(duì)脂肪間充質(zhì)干細(xì)胞的生物學(xué)影響 目的：構(gòu)建出具有靶向治療功能的新型TGF-β3融合蛋白LAP-MMP-mTGF-β3,并通過慢病毒載體包裝后轉(zhuǎn)染脂肪間充質(zhì)干細(xì)胞(ADSCs),兩者培養(yǎng)在多肽水凝膠支架內(nèi),以驗(yàn)證其可行性與靶向特異性。 方法：消化法分離得到兔ADSCs,通過流式法鑒定ADSCs表面抗原及特殊染色法鑒定ADSCs多向分化能力。通過基因重組方法將LAP, mTGF-p3和基質(zhì)金屬蛋白酶(MMP)的酶切位點(diǎn)PLGLWA分別插入到真核質(zhì)粒表達(dá)載體GV287,得到重組TGF-P3融合蛋白LAP-MMP-mTGF-β3的質(zhì)粒。用慢病毒載體包裝質(zhì)粒后轉(zhuǎn)染ADSCs,并培養(yǎng)在PRG/TB多肽水凝膠內(nèi)。CCK8法檢測增殖細(xì)胞增殖,鈣黃綠素/碘化丙啶法檢測細(xì)胞在凝膠內(nèi)部的存活,Elisa法檢測MMP酶刺激后上清液中的mTGF-p3。 結(jié)果：成功分離培養(yǎng)出ADSCs,并且ADSCs培養(yǎng)在PRG/TB混合多肽水凝膠內(nèi)可以提高增殖。慢病毒載體及PRG/TB多肽水凝膠對(duì)ADSCs的毒副作用小,并且MMP酶存在時(shí)可激活重組TGF-3融合蛋白釋放活性的TGF-β3。 結(jié)論：成功構(gòu)建出慢病毒載體包裝的新型融合蛋白LAP-MMP-mTGF-β3,慢病毒載體包裝的新型融合蛋白轉(zhuǎn)染的ADSCs與PRG/TB多肽水凝膠一起具有靶向性修復(fù)軟骨缺損的應(yīng)用前景。 第四部分LV-mTGF-p3轉(zhuǎn)染的脂肪間充質(zhì)干細(xì)胞在雙親多肽水凝膠內(nèi)誘導(dǎo)成軟骨細(xì)胞的研究 目的：探討功能性自組裝PRG/TB水凝膠支架與慢病毒包裝的融合蛋白LAP-MMP-mTGF-β3轉(zhuǎn)染的兔脂肪間充質(zhì)干細(xì)胞(ADSCs)誘導(dǎo)成軟骨的影響。 方法：將慢病毒包裝的融合蛋白LAP-MMP-mTGF-β3轉(zhuǎn)染第三代ADSCs后,與功能性PRG/TB水凝膠支架共同培養(yǎng)在軟骨細(xì)胞誘導(dǎo)液中,并增加10ng/m)基質(zhì)金屬蛋白酶-1(MMP-1)促進(jìn)融合蛋白LAP-MMP-mTGF-p3釋放TGF-β3,促進(jìn)ADSCs分化成軟骨細(xì)胞。3天后用Western blot檢測TGF-P3在ADSCs中的表達(dá)。ADSCs誘導(dǎo)分化成軟骨細(xì)胞21天后,分別用Real-time PCR和Western blot檢測軟骨細(xì)胞特異性基因Aggrecan (ACAN),II型膠原(COL2A1)和SOX-9的mRNA和蛋白的表達(dá)。 結(jié)果：慢病毒包裝融合蛋白LAP-MMP-mTGF-β3轉(zhuǎn)染ADSCs效率能達(dá)到90%以上。3天后轉(zhuǎn)染慢病毒的ADSCs成功檢測到TGF-β3的表達(dá)。與未轉(zhuǎn)染融合蛋白LAP-MMP-mTGF-β3的ADSCs相比,轉(zhuǎn)染組明顯促進(jìn)了ADSCs向軟骨細(xì)胞分化。21天后轉(zhuǎn)染組與未轉(zhuǎn)染組相比,ACAN、COL2A1和SOX-9mRNA表達(dá)分別增加2.7,2.4和1.0倍,蛋白表達(dá)分別增加2.4,1.57和0.89倍。 結(jié)論：成功構(gòu)建的慢病毒包裝融合蛋白LAP-MMP-mTGF-p3轉(zhuǎn)染ADSCs可促進(jìn)ADSCs分化成軟骨細(xì)胞,具有很好的修復(fù)軟骨缺損的應(yīng)用前景。 第五部分功能性自組裝多肽水凝膠聯(lián)合LV-mTGF-β3轉(zhuǎn)染的ADSCs修復(fù)骨性關(guān)節(jié)炎的研究 目的：進(jìn)一步驗(yàn)證PRG/TB自組裝多肽水凝膠支架聯(lián)合LV-mTGF-β3轉(zhuǎn)染的兔脂肪間充質(zhì)干細(xì)胞(ADSCs)在體內(nèi)修復(fù)軟骨的能力。 方法：將慢病毒包裝的融合蛋白LAP-MMP-mTGF-β3轉(zhuǎn)染ADSCs后,與功能性PRG/TB自組裝多肽水凝膠支架共同培養(yǎng)在軟骨細(xì)胞誘導(dǎo)液中。21天后,在裸鼠背部分別種植不同組的ADSCs和多肽PRG/TB自組水凝膠混合物,觀察多肽凝膠的毒副作用,4W后注射部位的組織用阿利新藍(lán)染色和免疫組化染色檢測成軟骨的能力。新西蘭大白兔用注射MIA方法完成骨性關(guān)節(jié)炎造模,然后每七天注射一次多肽水凝膠和慢病毒轉(zhuǎn)染后的ADSCs混合物到骨性關(guān)節(jié)炎模型的膝關(guān)節(jié),注射PBS作為試驗(yàn)對(duì)照組。8W后,組織染色觀察多肽水凝膠聯(lián)合重組TGF-p3蛋白轉(zhuǎn)染的ADSCs修復(fù)關(guān)節(jié)的情況。 結(jié)果：慢病毒包裝的融合蛋白LAP-MMP-mTGF-β3轉(zhuǎn)染ADSCs組在裸鼠皮下生成的軟骨組織外形要大于其他組,阿利新藍(lán)染色和免疫組化染色陽性表達(dá)明顯高于其余組。注射多肽水凝膠和慢病毒轉(zhuǎn)染后的ADSCs混合物組的病理染色結(jié)果顯示明顯改善了兔膝關(guān)節(jié)OA中軟骨組織的退化。 結(jié)論：功能性自組裝多肽水凝膠聯(lián)合慢病毒包裝的重組TGF-β3蛋白轉(zhuǎn)染的ADSCs能夠在體內(nèi)生成軟骨組織,并明顯改善OA中軟骨組織退化的情況,具有很好的修復(fù)軟骨缺損的應(yīng)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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R684

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本文編號(hào)：1816443