用BrdU标记滞留细胞联合Sox2表达检测晶状体干细胞

Using Sox-2 expression-combined BrdU label-retaining to detect lens stem cells

目的检测小鼠晶状体上皮细胞中可能存在的晶状体干细胞及其分布位置。方法 60只(60眼)Balb/c小鼠随机分为BrdU标记组和生理盐水对照组,每组各30只(30眼)。BrdU标记组以150mg·kg-1剂量的10g·L-1BrdU每天2次腹腔注射,连续注射10d。生理盐水对照组以15mL·kg-1生理盐水每天2次腹腔注射,连续注射10d。在BrdU或生理盐水注射结束后0周、1周、2周、3周、4周、6周、8周、10周、12和14周分别处死3只小鼠,制备眼球冰冻切片。通过免疫荧光化学染色观察晶状体上皮BrdU标记细胞的量和分布变化。在BrdU标记组,通过免疫荧光化学染色观察0周、4周、8周、12和14周晶状体上皮细胞Sox2蛋白的表达及分布规律。通过BrdU-Sox2双标记免疫荧光染色观察BrdU-Sox2双标阳性细胞。结果 BrdU标记组在完成BrdU标记时(0周),晶状体上皮中央区和赤道区BrdU标记率均达100%;随着时间延长,中央区和赤道区的BrdU标记率逐渐下降,至12周时降至最低,中央区和赤道区分别为14.23%、5.86%(P=0.000);14周时中央区和赤道区分别为14.97%、6.11%(P=0.000),分别与12周时两区比较,差异均无统计学意义(均为P>0.05)。0周、4周、8周、12周和14周时,中央区Sox2阳性细胞率分别为3.74%、3.47%、4.23%、4.09%和3.70%,赤道区Sox2阳性细胞率分别为0.66%、0.37%、0.47%、0.33%和0.51%,各时间点中央区与赤道区Sox2阳性细胞率差异均无统计学意义(均为P>0.05)。Sox2在晶状体上皮细胞内表达稳定,主要分布于中央区。部分BrdU阳性细胞表达Sox2,即BrdU-Sox2双标阳性细胞,集中分布于晶状体上皮中央区。结论表达Sox2的标记滞留细胞可能是晶状体干细胞,主要分布于晶状体上皮中央区。

Objective To detect lens stem cells and their locations in mouse lens epithelial cell. Methods A total of 60 Balb/c mice （60 eyes） were randomly and evenly divided into BrdU labeling group and normal saline group（ NS group）. A 10-day intra peritoneal injection of l0 g . L -1 BrdU（ 150 mg . kg -1 ） was performed on BrdU label group ,2 times per day. A 10-day intraperitoneal injection of normal saline （ 15 mL .kg-1） was performed on NS group, 2 times per day. Three mice were killed in each group at ten time-points：0 week,1 week,2 weeks,3 weeks,4 weeks,5 weeks,8 weeks, l0 weeks, 12 weeks and 14 weeks after the ending of intraperitoneal injection. Then eye ball frozen sections were made. Cell number and distribution changes of label-retaining ceils in lens epithelial cell were observed using immunofluorescence staining. Protein expression of Sox-2 and the distribution in BrdU labeling group at the lst,4th,7th,9th and 10th time-point were observed using immunofluorescence staining. BrdU-Sox2 double immunofluorescence staining was then conducted to detect BrdU-Sox2 double positive cells. Results At the 1st time-point,BrdU-labeling rate in central zone and e- quatorial zone of lens epithelial cell in BrdU labeling group were both 100%. BrdU-labe- ling rate in the two zones gradually decreased with time and they were at their lowest at the 9th time-point（ 14.23% and 5.86% , respectively ;P = 0.000 ）. At the 10th time-point, BrdU-labeling rate in the two zones were 14.97% and 6.11% ,respectively（P =0. 000）. BrdU-labeling rate in the two zones at the 10th time-point is not significantly different from those at the 9th time-point（both P 〉0.05）. At all the five time-points,Sox2 posi- tive rate in central zone were 3.74% ,3.47 % ,4.23% ,4.09% and 3.70% ,respectively; Sox2 positive rate in equatorial zone were 0.55% ,0.37% ,0. 47% ,0.33% and 0.51% , respectively. No significant difference was observed between Sox2 positive rate in the two zones at any time-point（ all P 〉 0.05 ）. Sox2 expression in lens epithelium was stable and mainly found in central zone. BrdU-Sox2 double positive cells were label-retai ning cells which showed Sox2 expression. They were mainly found in central zone. Conclusion BrdU-Sox2 double positive cells might be lens stem cells, which are mainly found in central zone of lens eoithelium.