發(fā)布時(shí)間：2018-05-15 13:36

  本文選題：EGR1蛋白 + 富含血小板血漿��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的研究EGR1對(duì)肌腱干細(xì)胞的誘導(dǎo)分化能力及EGR1、PRP相關(guān)生長因子對(duì)兔肩袖損傷后肌腱修復(fù)的影響。方法(1)采用酶消化法取新西蘭大白兔髕韌帶中間部分肌腱組織,經(jīng)過酶切處理及培養(yǎng)后分離、純化得到肌腱干細(xì)胞(TSCs),之后免疫熒光染色檢測4,6-二脒基-2-苯基吲哚(DAPI)、TNMD抗體和SCX抗體并在熒光顯微鏡下觀察進(jìn)一步證實(shí)鑒定TSCs細(xì)胞。(2)用帶有表達(dá)EGR1基因的質(zhì)粒(pCDNA-EGR1)轉(zhuǎn)染實(shí)驗(yàn)組TSCs,用空白質(zhì)粒轉(zhuǎn)染對(duì)照組TSCs。應(yīng)用短片段發(fā)夾RNA(shRNA)lentiviral微粒靶向干擾TSCs中BMP12和Smad1基因的表達(dá)。在EGR1的作用下實(shí)驗(yàn)組和對(duì)照組的TSCs分別誘導(dǎo)培養(yǎng)14天向肌腱細(xì)胞分化,分離純化得到肌腱細(xì)胞,應(yīng)用免疫熒光染色和定量PCR對(duì)肌腱細(xì)胞進(jìn)行鑒定,并對(duì)其相關(guān)基因表達(dá)進(jìn)行檢測。(3)通過RNA提取與實(shí)時(shí)定量PCR檢測(qRT-PCR),在基因水平比較實(shí)驗(yàn)組和對(duì)照組TSCs分化生成肌腱細(xì)胞的能力,并在TSCs分化培養(yǎng)過程中(0h,12h,1d,3d,7d,14d)提取蛋白質(zhì)并應(yīng)用Western blotting檢測:通過BMP12/Smad1/5/8通路生成的蛋白產(chǎn)物并與正常肌腱愈合時(shí)產(chǎn)生這些產(chǎn)物作比較。(4)建立兔肩袖損傷模型(共40只),每只實(shí)驗(yàn)動(dòng)物切斷右側(cè)肩關(guān)節(jié)岡上肌肌腱,對(duì)左側(cè)肩關(guān)節(jié)行假手術(shù)處理形成對(duì)照組。損傷模型建立后6周行肩袖修補(bǔ)手術(shù),并將實(shí)驗(yàn)動(dòng)物分為三組(每組10只):A組,單純修補(bǔ)手術(shù);B組,修補(bǔ)手術(shù)+TSCs植入(肌腱損傷部位);C組,修補(bǔ)手術(shù)+EGR1-TSCs植入(肌腱損傷部位)+PRP注射(肌腱-骨界面)。肩袖修補(bǔ)手術(shù)后8周,處死所有組實(shí)驗(yàn)動(dòng)物,獲取每只實(shí)驗(yàn)動(dòng)物雙側(cè)肱骨大結(jié)節(jié)連同附著于其上的岡上肌肌腱。每組取出3分樣本用于mRNA提取和蛋白質(zhì)提取(用于Western blotting);每組中剩余組織樣本(包括取自手術(shù)修補(bǔ)部位的岡上肌肌腱及肱骨大結(jié)節(jié))制成5μm厚的石蠟切片,進(jìn)行HE染色、免疫組化分析及來評(píng)估I型膠原蛋白的形成情況,評(píng)價(jià)肌腱的愈合情況。結(jié)果(1)TSCs肌腱干細(xì)胞培養(yǎng)成功,DAPI標(biāo)記的第三代TSCs細(xì)胞核可見明亮藍(lán)色熒光(圖3),標(biāo)記率達(dá)100%,細(xì)胞活性較高。NMD、SCX染色細(xì)胞質(zhì)呈綠色熒光,可觀察細(xì)胞整體形態(tài)及分布狀況良好。(2)實(shí)驗(yàn)組TSCs表達(dá)EGR1基因,對(duì)照組TSCs無表達(dá)。短片段發(fā)夾RNA(shRNA)lentiviral微粒靶向干擾TSCs中分化成功,分離純化得到肌腱細(xì)胞,免疫熒光染色和定量PCR對(duì)肌腱細(xì)胞進(jìn)行鑒定顯示為肌腱細(xì)胞。(3)RNA與實(shí)時(shí)定量PCR檢測(qRT-PCR),顯示實(shí)驗(yàn)組TSCs分化生成肌腱細(xì)胞并應(yīng)用Western blotting檢測,得到BMP12/Smad1/5/8通路生成的蛋白產(chǎn)物并與正常細(xì)胞相同。(4)兔肩袖損傷模型建立成功,HE染色、免疫組化分析均顯示I型膠原蛋白的形成情況良好,肌腱的愈合情況良好。結(jié)論EGR1對(duì)肌腱干細(xì)胞的誘導(dǎo)分化能力較好,EGR1、PRP相關(guān)生長因子對(duì)兔肩袖損傷后肌腱修復(fù)具有積極作用。
[Abstract]:Objective to study the ability of EGR1 to induce the differentiation of tendon stem cells and the effect of EGR1 / PRP-associated growth factor on tendon repair after rotator cuff injury in rabbits. Methods 1) the tendon of patellar ligament of New Zealand white rabbits was obtained by enzyme digestion. The tendon stem cells were purified and purified, and then immunofluorescence staining was used to detect the antibody to TNMD and SCX of DAPIN (4-diamidinyl-2-phenylindole) and to further confirm the identification of TSCs cells by fluorescence microscope.) the recombinant plasmid pCDNA-EGR1 was used to express the EGR1 gene. The experimental group was infected with TSCsand the control group was transfected with blank plasmid. Short hairpin RNA(shRNA)lentiviral particles were used to target BMP12 and Smad1 gene expression in TSCs. Under the action of EGR1, the TSCs of the experimental group and the control group were induced to differentiate into tendon cells for 14 days, and the tendon cells were isolated and purified. The tendon cells were identified by immunofluorescence staining and quantitative PCR. The expression of related genes was detected by RNA extraction and real-time quantitative PCR detection. The ability of TSCs to differentiate and form tendon cells was compared at the gene level between the experimental group and the control group. The protein was extracted and detected by Western blotting during TSCs differentiation and culture. The protein products produced by BMP12/Smad1/5/8 pathway were compared with those produced by normal tendon healing. 4) A rabbit rotator cuff injury model was established (40 rabbits, per unit). The tendon of the right supraspinatus muscle of the shoulder was amputated in only experimental animals. Sham operation was performed on the left shoulder joint to form a control group. Rotator cuff repair was performed 6 weeks after the establishment of the injury model. Experimental animals were divided into three groups: group A (n = 10), group B (n = 10) and group B (n = 10). TSCs implantation was performed in group C (site of tendon injury). Repair operation EGR1-TSCs implantation (site of tendon injury) PRP injection (tendon-bone interface). After 8 weeks of rotator cuff repair, all the experimental animals were killed, and the bilateral humeral tubercle and supraspinatus tendon attached to it were obtained. Three samples were taken from each group for mRNA extraction and protein extraction (for Western blotting). The remaining tissue samples from each group (including supraspinatus tendon and humeral tubercle taken from the site of surgical repair) were made into 5 渭 m thick paraffin sections for HE staining. Immunohistochemical analysis was used to evaluate the formation of type I collagen and the healing of tendons. Results Bright blue fluorescence could be seen in the third generation of TSCs nuclei labeled successfully by DAPI-labeled tendon stem cells (Fig. 3, the labeling rate was 100, and the cytoplasm stained with high activity. NMD-SCX was green fluorescence. The expression of EGR1 gene was observed in TSCs of experimental group and no expression of TSCs in control group. The short fragment hairpin RNA(shRNA)lentiviral particles were targeted to interfere with the differentiation of TSCs, and the tendon cells were isolated and purified. Immunofluorescence staining and quantitative PCR were used to identify the tendon cells as tendon cells. The results showed that the TSCs in the experimental group differentiated into tendon cells and was detected by Western blotting. The protein products produced by BMP12/Smad1/5/8 pathway and the same as normal cells were obtained. The rabbit rotator cuff injury model was successfully established by HE staining. The immunohistochemical analysis showed that the formation of type I collagen protein was good and the tendon healed well. Conclusion EGR1 can induce the differentiation of tendon stem cells. EGR1 / PRP-associated growth factor has a positive effect on tendon repair after rotator cuff injury in rabbits.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R686.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 Amir M Abtahi;Erin K Granger;Robert Z Tashjian;;Factors affecting healing after arthroscopic rotator cuff repair[J];World Journal of Orthopedics;2015年02期

2 張辛;敖英芳;;核心結(jié)合因子過表達(dá)定向誘導(dǎo)脂肪組織來源干細(xì)胞體內(nèi)、外成骨[J];中國運(yùn)動(dòng)醫(yī)學(xué)雜志;2013年12期

3 徐海棟;陳勇;盧俊浩;趙建寧;;帶線錨釘Krackow縫合法修復(fù)急性跟腱斷裂臨床研究[J];醫(yī)學(xué)研究生學(xué)報(bào);2013年02期

4 付國建;靳安民;張力;李森;王鵬程;許勇;趙衛(wèi)東;;rhBMP-2對(duì)兔肩袖損傷重建術(shù)后腱-骨愈合的組織學(xué)及生物力學(xué)研究[J];中國矯形外科雜志;2010年10期

5 汪洋;;肩袖損傷的研究概況[J];井岡山醫(yī)專學(xué)報(bào);2008年01期

6 童建軍,肖德明;肩袖損傷的形態(tài)學(xué)研究進(jìn)展及其臨床意義[J];中國矯形外科雜志;2005年14期

7 陳疾忤,陳世益,張鵬;肩袖間隙的解剖學(xué)研究及其臨床意義[J];中國臨床解剖學(xué)雜志;2004年01期

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