本文選題：組織工程 + 軟骨細(xì)胞　； 參考：《第四軍醫(yī)大學(xué)》2008年碩士論文

【摘要】： 軟骨缺損的修復(fù)是臨床一大難題,由于自體軟骨移植來源有限,組織工程化軟骨移植將是解決損傷軟骨修復(fù)的重要途徑,然而,組織工程化軟骨具有免疫原性。本課題擬通過轉(zhuǎn)染吲哚胺2,3-雙加氧酶(indoleamine 2,3-dioxygenase, IDO)基因以達(dá)到改變軟骨細(xì)胞移植后局部T細(xì)胞免疫應(yīng)答負(fù)調(diào)的目的,誘導(dǎo)移植受體的T細(xì)胞對同種異體組織工程化軟骨移植物的免疫耐受,為發(fā)展轉(zhuǎn)基因方法預(yù)防臨床同種異體移植后自身免疫排斥的技術(shù)奠定基礎(chǔ),實(shí)現(xiàn)組織工程化的種子細(xì)胞能用于不同免疫遺傳背景的患者,克服MHC(major histocompatibility complex,主要組織相容性復(fù)合物)的多態(tài)性和多基因性所形成的同種異體間的免疫屏障,達(dá)到組織工程化種子細(xì)胞的“通用化”,開辟了從移植物局部解決免疫排斥的新途徑。主要內(nèi)容及結(jié)果如下: (一)小鼠軟骨細(xì)胞的分離和培養(yǎng) 1.C57小鼠關(guān)節(jié)軟骨的獲取:體外經(jīng)0.2%Ⅱ型膠原酶37℃消化3～4 h后離心,收集可以獲取供實(shí)驗(yàn)用的大量軟骨細(xì)胞,臺盼藍(lán)染色消化法獲取的軟骨細(xì)胞活力在95%以上。 2.原代培養(yǎng)的軟骨細(xì)胞,MTT法檢測軟骨細(xì)胞生長曲線,發(fā)現(xiàn)軟骨細(xì)胞貼壁后于第2～5 d進(jìn)入指數(shù)生長期,7 d左右可以鋪滿瓶底,但是隨著細(xì)胞傳代次數(shù)的增加,細(xì)胞形成一代的時間明顯延長。 3.軟骨細(xì)胞分泌的細(xì)胞外基質(zhì),對甲苯胺藍(lán)染色呈明顯藍(lán)色異染反應(yīng),本實(shí)驗(yàn)獲取的軟骨細(xì)胞經(jīng)甲苯胺藍(lán)染色后,發(fā)現(xiàn)細(xì)胞純度在85%以上,但是隨著傳代次數(shù)增多軟骨細(xì)胞分泌細(xì)胞外基質(zhì)的能力下降,因此,選擇第2、3代軟骨細(xì)胞是用于實(shí)驗(yàn)的最佳時期。 (二)軟骨細(xì)胞pEGFP-N1-IDO質(zhì)粒轉(zhuǎn)染及基因表達(dá) 1.在最優(yōu)化的脂質(zhì)體和質(zhì)粒比例下(2:1),質(zhì)粒pEGFP-N1-IDO被脂質(zhì)體轉(zhuǎn)染至原代培養(yǎng)的軟骨細(xì)胞,轉(zhuǎn)染后的軟骨細(xì)胞仍能貼壁生長。 2.熒光顯微鏡和激光共聚焦顯微鏡對轉(zhuǎn)染后的軟骨細(xì)胞中EGFP表達(dá)的監(jiān)測表明,綠色熒光均勻地分布于整個細(xì)胞內(nèi),于轉(zhuǎn)染后12 h開始并在48 h時有較強(qiáng)表達(dá)。EGFP在胞漿和胞核中均有表達(dá),但胞漿中表達(dá)明顯高于胞核表達(dá)。 3.于轉(zhuǎn)染后不同時間點(diǎn)收集軟骨細(xì)胞,多聚甲醛固定后,流式細(xì)胞術(shù)檢測軟骨細(xì)胞中EGFP的表達(dá),轉(zhuǎn)染后隨著時間的延長,轉(zhuǎn)染效率呈先上升,后下降的趨勢。于轉(zhuǎn)染后24 h達(dá)到最高峰,48 h后轉(zhuǎn)染效率可高達(dá)36%。 4.軟骨細(xì)胞基因轉(zhuǎn)染48 h后,RT-PCR在RNA水平可檢測到mIDO表達(dá),與β-actin的光密度值相比,軟骨細(xì)胞中基因的相對表達(dá)量穩(wěn)定。 (三) mIDO轉(zhuǎn)染后軟骨細(xì)胞體外對T淋巴細(xì)胞增殖的影響 1.利用高壓液相色譜(HPLC)分析mIDO轉(zhuǎn)染軟骨細(xì)胞培養(yǎng)液中色氨酸的含量,24 h后單純軟骨細(xì)胞組色氨酸含量為4.93～5.71 mg/L,平均為5.3±0.39 mg/L,而基因轉(zhuǎn)染軟骨細(xì)胞組未檢測到色氨酸。在細(xì)胞培養(yǎng)液中色氨酸的降解,同時也伴隨著犬尿氨酸代謝產(chǎn)物的增加,對細(xì)胞上清液中色氨酸消除動力學(xué)研究表明,轉(zhuǎn)基因軟骨細(xì)胞上清液中色氨酸的降解明顯快于單純軟骨細(xì)胞組。 2.轉(zhuǎn)基因軟骨細(xì)胞體外混合淋巴細(xì)胞反應(yīng),以2×105個經(jīng)3 000 radγ射線照射的脾淋巴細(xì)胞作為抗原遞呈細(xì)胞和3×105個淋巴結(jié)淋巴細(xì)胞作為免疫應(yīng)答細(xì)胞,于混合反應(yīng)后第5 d、6 d、7 d、8 d通過MTT法檢測各組淋巴細(xì)胞增殖情況。結(jié)果顯示,較單純淋巴細(xì)胞脾細(xì)胞組,轉(zhuǎn)基因和未轉(zhuǎn)基因組淋巴細(xì)胞均出現(xiàn)了增殖(P0.02),且轉(zhuǎn)IDO基因組淋巴細(xì)胞增殖較未轉(zhuǎn)基因組明顯減慢(P0.05)。 3.混合淋巴細(xì)胞反應(yīng)中以CFSE標(biāo)記免疫應(yīng)答細(xì)胞,共同培養(yǎng)96 h后經(jīng)流式細(xì)胞術(shù)檢測應(yīng)答T細(xì)胞的增殖,轉(zhuǎn)基因組和未轉(zhuǎn)基因組應(yīng)答細(xì)胞均出現(xiàn)了增殖(P0.01),共形成9代細(xì)胞。IDO基因轉(zhuǎn)染組96 h后總體增殖指數(shù)為1.122±0.017,單純淋巴細(xì)胞陰性對照組為1.026±0.016,陽性單純軟骨細(xì)胞組為1.201±0.026,較陽性對照組,IDO基因組同種異體T淋巴細(xì)胞增殖得到明顯抑制。 結(jié)論:消化法可獲取供實(shí)驗(yàn)用的大量原代培養(yǎng)的軟骨細(xì)胞,且第2、3代軟骨細(xì)胞是用于實(shí)驗(yàn)的最佳時期;脂質(zhì)體有望成為原代培養(yǎng)軟骨細(xì)胞良好的基因轉(zhuǎn)導(dǎo)載體,mIDO被導(dǎo)入原代培養(yǎng)的軟骨細(xì)胞后,對EGFP監(jiān)測表明,IDO在細(xì)胞中得到了穩(wěn)定表達(dá),且主要表達(dá)于胞漿;轉(zhuǎn)基因細(xì)胞體外培養(yǎng)中明顯降解培養(yǎng)液中色氨酸濃度,為IDO通過降解局部微環(huán)境中色氨酸濃度而抑制同種異體T細(xì)胞增殖提供科學(xué)依據(jù);體外混合淋巴細(xì)胞反應(yīng)中,表達(dá)mIDO轉(zhuǎn)染軟骨細(xì)胞明顯抑制同種異體T細(xì)胞增殖,為基因轉(zhuǎn)染后軟骨細(xì)胞通過降解局部微環(huán)境中色氨酸的濃度抑制T細(xì)胞的增殖從而延長移植物的存活時間提供了思路。
[Abstract]:The repair of cartilage defects is a major problem in clinical practice. Due to the limited source of autologous cartilage transplantation, tissue engineered cartilage transplantation will be an important way to solve the repair of damaged cartilage. However, tissue engineered cartilage has immunogenicity. This topic is to be transfected through the transfection of indolamine 2,3- dioxygenase (indoleamine 2,3-dioxygenase, IDO) gene to achieve the goal. The aim of changing the negative modulation of the local T cell immune response after the transplantation of chondrocytes is to induce the immune tolerance of the transplant recipient's T cells to the allograft tissue engineered cartilage graft, which lays the foundation for the development of the transgenic method to prevent the autoimmune rejection after the allograft transplantation, and the tissue engineered seed cells can be used for the application. Patients with different immune genetic backgrounds have overcome the polymorphism of the MHC (major histocompatibility complex, the major histocompatibility complex) and the immune barrier formed by multiple genotypes to achieve the "generality" of the tissue engineered seed cells, and open up a new way to solve the immune rejection locally from the graft. The contents and results are as follows:
(1) isolation and culture of murine chondrocytes
The acquisition of articular cartilage in 1.C57 mice: centrifugation after digestion of 0.2% type of collagenase at 37 C for 3~4 h in vitro, a large number of chondrocytes for experimental use were collected. The vitality of chondrocytes obtained by trypan blue staining method was more than 95%.
2. primary cultured chondrocytes, MTT method was used to detect the growth curve of chondrocytes. It was found that the chondrocytes entered the exponential growth period from second to 5 d after the chondrocytes adhered to the wall, and it could be filled with the bottom of the bottle at about 7 d, but the time of cell formation was obviously prolonged with the increase of the number of cells.
3. the extracellular matrix secreted by chondrocytes showed a clear blue staining reaction to toluidine blue. The chondrocytes obtained by this experiment were stained with toluidine blue, and the purity of the cells was above 85%. However, with the increase of the number of cells, the capacity of the cartilage cells to secrete the extracellular matrix was decreased. Therefore, the selection of the chondrocytes of the 2,3 generation was used as a real one. The best time for testing.
(two) transfection and gene expression of pEGFP-N1-IDO plasmid in chondrocytes
1. under the optimal proportion of liposomes and plasmids (2:1), plasmid pEGFP-N1-IDO was transfected into the primary cultured chondrocytes by liposomes, and the chondrocytes after transfection could still grow on the wall.
The monitoring of the expression of EGFP in the transfected chondrocytes by 2. fluorescence microscopy and confocal microscopy showed that the green fluorescence was evenly distributed throughout the whole cell. The expression of.EGFP in the cytoplasm and nucleus was expressed at 12 h after transfection and at 48 h, but the expression in the cytoplasm was obviously higher than that of the nucleus.
3. after transfection, the chondrocytes were collected at different time points. After the polyformaldehyde was fixed, the expression of EGFP in cartilage cells was detected by flow cytometry. After the transfection, the transfection efficiency increased first and then decreased. After transfection, the highest peak was reached by 24 h, and the transfection efficiency could reach 36%. after 48 h transfection.
After transfection of 4. chondrocyte gene to 48 h, the expression of mIDO was detected at the level of RNA, and the relative expression of gene in cartilage cells was stable compared with the light density value of beta -actin.
(three) effects of chondrocytes transfected with mIDO on proliferation of T lymphocytes in vitro
1. the content of tryptophan in chondrocyte culture fluid transfected by mIDO was analyzed by high pressure liquid chromatography (HPLC). After 24 h, the content of tryptophan in pure chondrocyte group was 4.93 ~ 5.71 mg/L, with an average of 5.3 0.39 mg/L, while the gene transfected chondrocyte group was not detected by tryptophan. The study on the removal of tryptophan in cell supernatant by the increase of acid metabolites showed that the degradation of tryptophan in the supernatant of the transgenic chondrocytes was significantly faster than that in the simple chondrocyte group.
2. transgenic chondrocytes were mixed lymphocyte reaction in vitro, with 2 x 105 splenic lymphocytes irradiated by 3000 rad gamma rays as antigen presenting cells and 3 x 105 lymph node lymphocytes as immune response cells. After the mixed reaction, fifth D, 6 D, 7 d, 8 d were detected by MTT method. The results showed that the lymphocyte proliferation was more simple. In lymphocyte splenocytes group, proliferation (P0.02) was found in both transgenic and non transgenomic lymphocytes, and the proliferation of IDO genome was significantly slower than that of the non transgenome genome (P0.05).
3. the immune response cells were labeled with CFSE in the mixed lymphocyte reaction, and the proliferation of T cells was detected by flow cytometry after 96 h co culture. The proliferation of T cells was detected in both the transgenic and non transgenomic response cells (P0.01), and the total body proliferation index of the 9 generation cell.IDO gene transfected group was 1.122 + 0.017 after 96 h, and the simple lymphocyte negative was negative. The control group was 1.026 + 0.016, and the positive chondrocyte group was 1.201 + 0.026. Compared with the positive control group, the proliferation of IDO T cells was significantly inhibited.
Conclusion: the digestion method can obtain a large number of primary cultured chondrocytes for experimental use, and the 2,3 generation chondrocytes are the best time for the experiment. Liposome is expected to be a good carrier of gene transduction in the primary cultured chondrocytes. MIDO is introduced into the primary cultured chondrocytes. The monitoring of EGFP shows that IDO has obtained a stable table in the cell. The concentration of tryptophan in the culture medium in vitro culture of transgenic cells provides a scientific basis for IDO to inhibit the proliferation of allogenic T cells by degrading the concentration of tryptophan in the local microenvironment. In the mixed lymphocyte reaction in vitro, the expression of mIDO transfected to chondrocytes inhibits the increase of allogenic T cells. Colonization, after gene transfection, chondrocytes can inhibit the proliferation of T cells by degrading the concentration of tryptophan in local microenvironment and thus prolonging the survival time of the grafts.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R318.0;R392.4

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