EGR1、PRP相关生长因子对兔肩袖损伤后肌腱修复的影响

发布时间：2018-05-15 13:36

本文选题：EGR1蛋白 + 富含血小板血浆　；参考：《青岛大学》2017年硕士论文

【摘要】：目的研究EGR1对肌腱干细胞的诱导分化能力及EGR1、PRP相关生长因子对兔肩袖损伤后肌腱修复的影响。方法(1)采用酶消化法取新西兰大白兔髌韧带中间部分肌腱组织,经过酶切处理及培养后分离、纯化得到肌腱干细胞(TSCs),之后免疫荧光染色检测4,6-二脒基-2-苯基吲哚(DAPI)、TNMD抗体和SCX抗体并在荧光显微镜下观察进一步证实鉴定TSCs细胞。(2)用带有表达EGR1基因的质粒(pCDNA-EGR1)转染实验组TSCs,用空白质粒转染对照组TSCs。应用短片段发夹RNA(shRNA)lentiviral微粒靶向干扰TSCs中BMP12和Smad1基因的表达。在EGR1的作用下实验组和对照组的TSCs分别诱导培养14天向肌腱细胞分化,分离纯化得到肌腱细胞,应用免疫荧光染色和定量PCR对肌腱细胞进行鉴定,并对其相关基因表达进行检测。(3)通过RNA提取与实时定量PCR检测(qRT-PCR),在基因水平比较实验组和对照组TSCs分化生成肌腱细胞的能力,并在TSCs分化培养过程中(0h,12h,1d,3d,7d,14d)提取蛋白质并应用Western blotting检测:通过BMP12/Smad1/5/8通路生成的蛋白产物并与正常肌腱愈合时产生这些产物作比较。(4)建立兔肩袖损伤模型(共40只),每只实验动物切断右侧肩关节冈上肌肌腱,对左侧肩关节行假手术处理形成对照组。损伤模型建立后6周行肩袖修补手术,并将实验动物分为三组(每组10只):A组,单纯修补手术;B组,修补手术+TSCs植入(肌腱损伤部位);C组,修补手术+EGR1-TSCs植入(肌腱损伤部位)+PRP注射(肌腱-骨界面)。肩袖修补手术后8周,处死所有组实验动物,获取每只实验动物双侧肱骨大结节连同附着于其上的冈上肌肌腱。每组取出3分样本用于mRNA提取和蛋白质提取(用于Western blotting);每组中剩余组织样本(包括取自手术修补部位的冈上肌肌腱及肱骨大结节)制成5μm厚的石蜡切片,进行HE染色、免疫组化分析及来评估I型胶原蛋白的形成情况,评价肌腱的愈合情况。结果(1)TSCs肌腱干细胞培养成功,DAPI标记的第三代TSCs细胞核可见明亮蓝色荧光(图3),标记率达100%,细胞活性较高。NMD、SCX染色细胞质呈绿色荧光,可观察细胞整体形态及分布状况良好。(2)实验组TSCs表达EGR1基因,对照组TSCs无表达。短片段发夹RNA(shRNA)lentiviral微粒靶向干扰TSCs中分化成功,分离纯化得到肌腱细胞,免疫荧光染色和定量PCR对肌腱细胞进行鉴定显示为肌腱细胞。(3)RNA与实时定量PCR检测(qRT-PCR),显示实验组TSCs分化生成肌腱细胞并应用Western blotting检测,得到BMP12/Smad1/5/8通路生成的蛋白产物并与正常细胞相同。(4)兔肩袖损伤模型建立成功,HE染色、免疫组化分析均显示I型胶原蛋白的形成情况良好,肌腱的愈合情况良好。结论EGR1对肌腱干细胞的诱导分化能力较好,EGR1、PRP相关生长因子对兔肩袖损伤后肌腱修复具有积极作用。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R686.1

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