| Objective： To explore the mechanism of17β-estradiol inhibitingapoptosis of rat nucleus pulposus cells(NPCs) via integrin subtype α1β1and/orα2β1.Methods：Rat NPCs were primarily cultured by methods of enzymedigestion, and subcultured to next generation by digestion of0.2%typsincontaining EDTA when cells were confluent. NPCs of the passage3(P3) werecultured in complete DMEM/F12without fetal bovine serum(FBS) andphenol red, and then experiments were performed in group A, B, and C, sixsamples in each. Group A acted as a control with a little ethanol added into.17β-estradiol was added into group B while17β-estradiol and ICI182780ingroup C.48hours later, NPCs were identified by immunocytochemistry ofcollagen II; cell apoptosis was determined by flow cytometry(FACS) andTUNEL assay; cell attachment to collagen I and II was detected by celladhesion assay, and integrin expression level was determined by westernblotting.Results:(1)The first-passage NPCs presented polygonal and/or longfusiform, with clear outline. The nuclei were round or oval. Secretory granulescan be seen in the cytoplasm. NPCs began to adhere to the bottom of cultureflask in24or48hours, secreted type II collagen and grew into logarithmicphase in7or8days. Generally, NPCs can be subcultured to6-8passageslifelong.(2)After48hours of culture, immunocytochemistry displayed thatcellular purity of NPCs was of high level with positive type II collagen.(3)Cell apoptotic incidence was effectively decreased by estrogen via TUNELassay. FACS revealed as follows: cell apoptosis in early stage was4.00%0.16%in control group,0.41%0.19%in estrogen group, and3.20%0.05%in estrogen+ICI group, respectively; cell apoptosis in advanced stage was2.01%0.18%in control group,0.50%0.10%in estrogen group,2.63%0.20%in estrogen+ICI group, respectively. Data difference betweengroups was statistically significant(F=24.20，P<0.001). The apoptotic rate inestrogen group was lower than that of control group (P<0.01) andestrogen+ICI group (P<0.01). Difference of apoptotic rate between controlgroup and estrogen+ICI group was no statistically significance.(4) Celladhesion to type II collagen showed that OD value was0.600.03，95%CI(0.53,0.68) in control group,0.730.04，95%CI(0.63,0.83) in estrogengroup,0.550.07，95%CI(0.37,0.73) in estrogen+ICI group, respectively. Celladhesion to collagen I showed no statistical difference between groups, whilethat to collagen II exhibited statistical difference (F=10.68, P<0.05). OD valuein estrogen group was significantly higher than that both in control group(P<0.05) and estrogen+ICI group (P<0.001). Data difference between controlgroup and estrogen+ICI group was no statistically significance.(5)Westernblotting of integrin α2subunit demonstrated that grey value（/β-actin） was0.230.005，95%CI (0.22,0.24) in control group,0.510.019，95%CI (0.46,0.55) in estrogen group,0.210.009，95%CI (0.18,0.23) in estrogen+ICIgroup, respectively. In addition, western blot of integrin β1subunitdemonstrated that grey value（/β-actin）was0.260.011，95%CI (0.24,0.29)in control group,0.500.031，95%CI (0.43,0.58) in estrogen group,0.250.018，95%CI (0.20,0.29) in estrogen+ICI group, respectively. Westernblotting revealed, integrin α2β1, not α1β1, was significantly upregulated++by17β-estradiol (P<0.01).Conclusions:17β-estradiol protects rat nucleus pulposus cells fromapoptosis, probably by upregulating integrin α2β1and then enhancing cellattachment to collagen type II. |
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