Experimental Studies Of Rat Lens Regeneration Model In Stem Cell-like Cells And Their Associated Signaling Pathways

Objective:To modify the model of rat lens regeneration, and observe this phenomenon dynamicly to verify the function and position of lens stem cell-like cells in the process of lens regeneration. On the basis of this lens regeneration model in vivo, we would further research whether such signal pathway related to stem cells as Notch signaling was involved in the conversion of lens stem cell-like cells to lens epithelial cells.Methods:The right eyes of 6-8 week Wistar rats were performed extracapsular lens extraction through transparent corneal incision. An arc incision of 1/3 quadrant in the anterior capsule was performed instead of continuous circular capsulorhexis. The regeneration of rat lens was observed by slit-lamp and histopathology immediately after surgery and one day, three days, one week, two weeks, and one month postoperatively respectively. Real-time quantitative polymerase chain reaction (RTQ-PCR) was performed to detect the expression of ABCG2, Nestin, PCNA and Telomerase, which was usually accepted as stem cell markers and used to verify the existence of tissue stem cells. Furtherly, the capsule was devided into two parts:the anterior capsule and the posterior capsule which involved the equator part. RTQ-PCR was performed to orientate the position of lens stem cell-like cells by detecting different expression of ABCG2, Nestin, PCNA and Telomerase. Immunohistochemistry was used to detect the expression of ABCG2 and PCNA, which furtherly verify the position of lens stem cell-like cells in paraffin sections. BrdU was injected intraperitoneally (twice daily) three days before surgery. After surgery, eyes were removed and precessed for immunohistochemistry at different check time. Meanwhile, RTQ-PCR was used to detect the expression of Notch 1, Notch2, Jagged 1 and CyclinD1. Western blot was also performed to detect the levels of their expression in different time after surgery.Results:All eyes exhibited clinically evident lens regeneration 1 week after surgery. One month later, new regenerated lens was apparent and transparent in all rats, except for the opacity part near the incision. The farer away the anterion incision, the regenerated lens exhibited more transparent. Historically, lens epithelial cells were left behind under the anterior and equator capsule immediately after extracapsular lens extraction. The capsualr bag was open and no contact between the anterior and posterior lens capsule occurred. One day after surgery, some epithelial cells proliferated in the equator part,a monolayer of LECs covered the inner surgace of the capsular bag. Three days after surgery, the capsular bag was filled with proliferated lens epithelial cells and the anterior capsule at the incision margin rolled upwards with some epithelial cell proliferated. On the seventh day, lens fibers occurred in the middle of capsular bag, but the nuclears were still near the posterior capsules. On the second week, new lens fibers continued to increase and the nuclei of LECs lining the posterior capsule miagrated away from the basement membrane. One month later, the capsule was full of differentiated lens fibers with an established equator with well differentiated bow regions. While in the anterior incision, the anterior and posterior capsule contacted and more and more lens epithelial cells proliferated. Regenerated lens was divided into two parts apart, which had integrated capsule with anterior, posterior and equator part respectively.To survey the expression of tissue stem cells and their related signal pathway, the mRNA of ABCG2/PCNA/Nestin/Telomerase and Notch1/Notch2/Jaggedl/CyclinDl were detected by RTQ-PCR. At any time point, the mRNA of these markers could be found and its relative expression enhanced early after surgery. Immediately after surgery, the capsule was divided into the anterior part and the posterior part which involved the equator. All mRNA of four stem cell markers were detected in the two parts by RTQ-PCR.Immunohistochemistry shows that after extracapsular lens extraction, residual lens epithelial cells both in the anterior and the equator part could proliferate and expressed ABCG2 and PCNA positive regions. BrdU incorporation experiment also showed that following surgery, the resident LECs both in the anterior and equator region incorporated BrdU, indicating that these cells were stem cell-like cells and choud be recurited to proliferate.Western blot shows that during lens regeneration, Notch2 appeared in the early period after surgery, subsequently, Jagl and Notch1 also played a role in lens stem cell-like cells proliferation and differentiation.Conclusions:After extracapsular lens extraction, new lens soon regenerated in rats.This process was similar to nornal lens development. The regenerated lens appeared more transparent while the capsule was more intact. However lens epithelial cells proliferated abnormally near the anterior incision, which appeared evident opacity without lens fiber formed historically. During lens regeneration, tissue stem cell-like cells played an improtant role in it, which was verified to be existed both in the anterior and the equator part of the capsule. Our new model of lens regeneration also verified the role of Notch signal pathway in proliferation and differentation of lens stem cell-like cells.