大鼠晶状体再生模型中干细胞样细胞及其相关信号通路的实验研究

发布时间：2018-02-04 17:51

本文关键词： 大鼠再生晶状体干细胞信号通路　出处：《青岛大学》2012年博士论文　论文类型：学位论文

【摘要】：目的：改进大鼠晶状体再生模型,观察晶状体再生过程中干细胞标志物的表达,并初步探讨Notch信号通路在晶状体干细胞样细胞增殖分化中的作用。方法：第一部分：大鼠晶状体再生模型的改进对6-8周成年健康Wistar大鼠一眼采用透明角膜切口行晶状体囊外摘除术,术中不采用环形撕囊,而只将晶状体前囊膜做1/3象限的弧形切开,分离出晶状体。术后即刻、1天、3天、1周、2周、1月,分别裂隙灯下观察晶状体再生情况并照相,HE染色行病理组织学检查。第二部分：晶状体再生过程中干细胞标记物的表达及定位 1.将晶状体囊外摘除术后3天、1周、2周及1月的晶状体再生组织分别取出,提取RNA并反转录为cDNA,然后通过荧光定量PCR的方法检测再生组织中干细胞标记物ABCG2、Nestin、PCNA及Telomerase的表达,确定再生晶状体组织中干细胞样细胞是否存在。设对侧未手术眼为正常对照。 2.晶状体囊外摘除术后即刻,将晶状体囊袋组织分为前囊及后囊(包含赤道部)两部分,分别提取RNA并通过荧光定量PCR的方法检测各部分干细胞标记物的表达,初步定位晶状体干细胞样细胞。 3.石蜡包埋的大鼠眼作厚度为4微米的石蜡切片,免疫组化检测ABCG2及PCNA的表达,对晶状体干细胞样细胞进一步定位。 4.晶状体囊外摘除术前3天每天两次注射BrdU,最后一次注射后行一眼的晶状体囊外摘除术,术后即刻、术后1天、3天及1周分别取眼球石蜡包埋后切片,通过免疫组化的方法检测BrdU的表达,确定增殖的细胞所在,辅助定位晶状体干细胞样细胞。第三部分：Notch信号通路与晶状体再生 1.将晶状体囊外摘除术后不同时间点的晶状体再生组织取出,提取RNA并反转录为cDNA,然后通过荧光定量PCR的方法检测Notch信号通路信号分子Notch1、Notch2及Jag1的表达,并且通过对细胞周期调控蛋白CyclinDl的PCR检测,初步验证此信号通路是否存在于晶状体再生过程中。 2.晶状体囊外摘除术后即刻、3天及1周,将再生晶状体组织取出,分别提取组织蛋白,通过Western blot的方法检测各部分组织中Notch1、Notch2及Jag1的表达及变化。结果：第一部分：大鼠晶状体再生模型的改进大鼠晶状体囊外摘除术后,所有手术眼均很快发生晶状体组织再生,其程度随时间推移逐渐明显。至术后1月,已形成典型的再生晶状体,其中远离囊膜切开处的再生晶状体透明度高,但囊膜切开处混浊明显。晶状体囊外摘除术后即刻,仅前囊膜下及赤道部残留少量单层晶状体上皮细胞(LEC)；术后1天,赤道部残留晶状体上皮细胞开始增生。术后3天,前后囊间的囊袋内布满增生的晶状体上皮细胞,前囊切开处两断端翻折伴晶状体上皮细胞异常增生。术后1周,囊袋内开始充满早期增生的晶状体纤维,但增生的晶状体上皮细胞仍紧贴后囊膜。术后2周,晶状体前囊及赤道部的上皮细胞明显增生,囊袋内的晶状纤维向前伸长呈带状,其细胞核已远离后囊膜；在前囊切开的地方,前囊弯曲皱折,细胞异常增生,前后囊贴合紧密处亦充满增生细胞,而两侧的晶状体囊袋相对完整,细胞增生分化排列规则,内侧前后囊结合处细胞增生分化类似正常晶体的赤道部,整个再生的晶状体类似葫芦状。术后1月,再生的晶状体纤维填充整个囊袋,赤道部形成典型的弓形带,与正常晶状体赤道部形态类似。而在前囊切开的部分,晶状体上皮细胞增生而无晶状体纤维形成,两侧的葫芦状再生晶状体排列规则,各自形成相对完整的前囊、赤道部以及填充囊袋的晶状体纤维。第二部分：晶状体再生过程中干细胞标志物的表达及定位荧光定量PCR的方法检测到正常晶状体及术后不同时间点的再生晶状体均有组织干细胞标记物信号的存在。晶状体囊外摘除术后即刻,将前囊及包含赤道部的后囊分开分别提取RNA后行PCR检测,两部分均有干细胞标记物信号的扩增。选取四种干细胞标记物中的两种PCNA及ABCG2为代表行组织的免疫组化检测。在晶状体再生过程中,晶状体前囊及赤道部均可检测到两种信号标记的存在,特别是前囊切开的部分,晶状体上皮细胞异常增生,而此处PCNA及ABCG2的表达明显增多。腹腔注射BrdU后建模,然后通过免疫组化的方法分别进行检测。结果显示,正常成熟晶状体组织中赤道部及前囊均散在少量BrdU阳性细胞,注药后1天即可检测到,术后1周仍可见。而在晶状体再生组织中,术后即刻赤道部及前囊即可见BrdU阳性细胞。术后1天,赤道部晶状体上皮细胞开始增生,BrdU阳性细胞亦开始增多,尽管前囊晶状体上皮细胞呈单层,但亦散在少量BrdU阳性细胞。术后3天,赤道部及前囊均即可见较多BrdU阳性细胞,特别是前囊切开的地方,晶状体上皮细胞异常增生,相应BrdU阳性细胞亦较多。术后1周时,赤道部及前囊仍可见BrdU阳性细胞,但较术后3天明显减少。第三部分：Notch信号通路在晶状体再生中的表达及作用正常晶状体组织及再生晶状体组织中均检测到Notch通路受体Notch1、Notch2及Notch配体Jag1的表达,且晶状体囊外摘除术后早期表达量呈上升趋势,术后1周表达量最高,之后逐渐降低。而相应的细胞周期调控关键蛋白CyclinD1的表达趋势与此类似。根据PCR结果,选取术后1周内的晶状体再生组织行Western blot检测,结果显示术后即刻即有Notch2的表达,术后3天Notch2表达明显,术后1周表达降低,而Notch1术后1周表达明显。Jag1在术后3天开始表达,术后1周表达量明显。但作为对照的正常晶状体组织未检测到各信号分子蛋白的表达。结论： 1改进的大鼠晶状体再生模型短期内即可再生出新的晶状体组织,且远离前囊切开的部分再生组织较透明,相应地晶状体上皮细胞排列较规则。而前囊切开部分的晶状体组织明显混浊,相应晶状体上皮细胞异常增生,未发生晶状体纤维化改变。 2正常晶状体及再生晶状体组织中均存在干细胞样细胞,其在晶状体再生过程中发挥重要作用。正常的晶状体组织内仅少量的干细胞样细胞存在,以维持晶状体的自我更新；但在晶状体损伤后的再生过程中,前囊以及赤道部均有干细胞样细胞发挥作用,从而迅速发生增殖分化,再生出新的晶状体组织。 3 Notch信号通路于晶状体再生的早期即被激活,调控晶状体干细胞样细胞的增殖。
[Abstract]:Purpose : To improve the rat lens regeneration model and to observe the expression of stem cell markers in lens regeneration , and to investigate the role of Notch signaling pathway in the proliferation and differentiation of lens stem cell - like cells . Method : In the first part , the rat lens regeneration model was improved for 6 - 8 weeks of adult healthy Wistar rats . Part 2 : Expression and localization of stem cell marker during lens regeneration 1 . The lens regenerating tissues were taken out , RNA was extracted and reverse transcribed into cDNA for 3 days , 1 week , 2 weeks and 1 month after the lens capsule extirpation , then the expression of ABCG2 , Nestin , PCNA and PCNA was detected by fluorescence quantitative PCR . 2 . The lens capsule was divided into two parts : the anterior capsule and the posterior capsule ( including the equatorial portion ) . RNA was extracted and the expression of the marker of each stem cell was detected by fluorescence quantitative PCR . The lens stem cell - like cells were initially located . 3 . Paraffin - embedded rat eyes were sectioned with paraffin sections with a thickness of 4 microns . The expression of ABCG2 and PCNA was detected by immunohistochemistry . 4 . After the lens capsule extirpation was performed twice daily for 3 days , the posterior lens capsule was injected twice a day , and then the posterior lens capsule was taken out after the last injection . The paraffin - embedded sections of the eyeball were taken respectively at 1 day , 3 days and 1 week after the operation , and the expression of the cells was detected by immunohistochemistry . The proliferation of the cells was determined , and the lens stem cells like cells were located in the auxiliary position . Part III : Notch signaling pathway and lens regeneration 1 . The lens regenerative tissues at different time points were taken out , RNA was extracted and reverse transcribed into cDNA , then the expression of Notch1 , Notch2 and Jag1 was detected by fluorescence quantitative PCR . 2 . Immediate , 3 - day and 1 - week post - cataract extraction , the tissue protein was extracted and the expression and change of Notch1 , Notch2 and Jag1 in each tissue were detected by Western blot . Results : Part I : Improvement of Rat Lens Regeneration Model The lens tissue regeneration was observed in all the eyes of the lens after cataract extraction in rats . The degree of lens tissue regeneration was gradually increased over time . By January , the lens was formed with a typical regeneration lens , in which the lens had a higher transparency than the regeneration lens at the incision of the capsule , but the opacity of the capsule was obvious . The lens epithelial cells of the anterior capsule and the equatorial portion of the lens were proliferated . The lens epithelial cells in the anterior capsule and the equatorial region were proliferated . The lens fibers of the anterior capsule and the anterior capsule were more complete . The lens fibers in the anterior and posterior capsule were more complete . The lens fibers in the anterior and posterior capsule were more complete . The fluorescence quantitative PCR method was used to detect the presence of marker signal of tissue stem cells in both the normal lens and the regenerated lens at different time points after operation . The anterior capsule and the posterior capsule containing the equatorial portion were separated from the posterior capsule containing the equatorial portion , and then the stem cell marker signal was amplified . Both PCNA and ABCG2 in four stem cell markers were selected to represent the immunohistochemical detection of the tissue . In the process of lens regeneration , both the anterior capsule and the equatorial portion of the lens could detect the existence of two signal markers , especially those of anterior capsule , and abnormal proliferation of lens epithelial cells , and the expression of PCNA and ABCG2 was significantly increased here . The results showed that both the equatorial region and the anterior capsule of the normal mature lens were scattered in a small number of cells . After 1 day of operation , the cells of the equatorial region and the anterior capsule could be seen . The expression of Notch1 , Notch2 and Notch ligand Jag1 was detected in both the normal lens tissue and the regeneration lens tissue , and the early expression level of the lens capsule was increased . The expression level was highest at 1 week postoperatively , and the expression of Cyclin D1 was similar to that of the corresponding cell cycle regulation key protein Cyclin D1 . According to the results of PCR , Western blot was used to detect the expression of Notch2 in 1 week after surgery . The expression of Notch2 was significantly lower in 3 days after operation . The expression of Notch2 was significantly decreased at 1 week after operation . Conclusion : 1 . The improved lens regeneration model can regenerate the new lens tissue in the short term , and the partial regeneration tissues away from the anterior capsule are more transparent , and the lens epithelial cells are arranged more regularly . There are stem cell - like cells in both normal and regenerative lens tissues . It plays an important role in lens regeneration . Only a small amount of stem cell - like cells exist in the normal lens tissues to maintain the self - renewal of the lens . However , in the regeneration process after lens injury , the stem cells - like cells play a role in the anterior capsule and the equatorial portion , thereby rapidly proliferating and differentiating and regenerating the new lens tissue . The 3 Notch signaling pathway is activated at the early stage of lens regeneration and regulates the proliferation of lens stem cell - like cells .

【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R779.6;R-332

【参考文献】