ABSTRACT
[Objective] To observe the effect of stanozolol on proliferation and differentiation of cultured growth plate chondrocytes in vitro.[Methods] At 3 week of age,Sprague–Dawley rats received 2.5 mg/kg in slow-released GnRHa (triptorelin) which was repeated every 2 weeks for 2 times,at 7-week old.The tibial growth plate cartilage were aseptically dissected and tripsin and EDTA digested for 0.5 h,then collagenase digested for 3 h at 37 ℃.Chondrocytes were cultured in DMEM:F12 medium for 48 h,the cells were starved for 24 h in serum-free DMEM:F12 medium before stanozolol treatment.In dose-effect groups,chondrocytes were incubated in serum-free media in various concentrations of stanozolol for 48 h.In time-course groups,chondrocytes were incubated in serum-free media in various times of stanozolol (10-8 mol/L).immunohistochemical staining of collagen Ⅱ,Ⅹ,PCNA,and MTT were conducted.[Results] The results of MTT,PCNA,and typeⅡcollagen synthesization demonstrated stanozolol enhanced the proliferation of the chondrocytes,time-course studies had shown that the proliferation were maximally stimulated by stanozolol after 2 or 3 days of incubation and decreased again after longer periods of incubation.Stanozolol stimulated the proliferation of the chondrocytes dose-dependently at 10-11 mol/L and 10-8 mol/L,maximally stimulatory concentrations of Stanozolol was 10-9 ～ 10-8 mol/L,and decreased again after higher concentration of stanozolol.Stanozolol did not stimulated type X collagen synthesization from 10-11 mol/L ～ 10-8 mol/L,but experiments showed that type X collagen was already stimulated after incubation in Stanozolol (10-7 ～ 10-5 mol/L).Time-course studies had shown those typeⅩcollagen synthesizations were stimulated by stanozolol after 4 ～ 5 days of incubation.[Conclusion] Stanozolol enhances the proliferation of chondrocytes of pubertal female rat treated with GnRHa in vitro (time-course- dependent and concentration -dependent).
ABSTRACT
Objective To investigate the possible mechanism of the effect of short-term gonadotropin-re-leasing hormone agonist(GnRHa)on linear growth in female pubertal rats. Methods Forty 3-week-old female rats were randomly divided into 5 groups(n=8 each). One group was sacrificed as base-line control. Group OVX was operated for ovariectomy at the beginning of experiment. Group Gn and group E2 each received two intramuscu-lar injections of 2.5mg·kg-1 triptorelin 2 weeks apart, and group E2 received additional daily 1μg·kg-1·d-1estradiol(E2)s. c. at three days after the second GnRHa injection for 11 days. Group Ctrl was sham-operated ascontrol. Each rat, except for the base-line control group. received 30mg/kg oxytetracycline s. c. and 20mg/kgcalcein s. c. 9 and 2 days respectively before sacrifice. Hepatic GH receptor mRNA, insulin-like growth factor(IGF)-I and IGF binding protein(IGFBP)-3, circulating IGF-I and IGFBP-3, local IGF-I/IGF-I receptor(IGF-IR)and proliferation rate(PFR)in epiphyseal growth plate(EGP)were evaluated after 4-week treatment.Results Similar to group OVX, the rats in group Gn became taller and heavier than group Ctrl with greater tibial length, wider EGP, greater longitudinal growth rate(LGR)and higher PFR(P<0.05 or P<0.01). Estrogen supplement reversed the effect of GnRHa. There was no statistical difference among the 4 groups regarding plasma IGF-I and IGFBP-3, hepatic IGF-I and IGFBP-3 mRNA levels, local IGF-I and IGF-I R levels in EGF. GnRHa down-regulated hepatic GHR mRNA expression, which was reversed by estrogen supplement. Conclusion GnRHa accelerates longitudinal growth of female pubertal rats. Estrogen deprivation contributes to GnRHa-induced alteration of linear growth in female rats, through improving PFR and suppressing the senescence of EGP. The underlying mechanism does not attribute to endocrine change of GH/IGF-I axis or local IGF-I/IGF-IR in EGP.
ABSTRACT
Objective To observe the effect of stanozolol(ST) on long bone growth and maturation of pubertal female rats treated with gonadotropin releasing hormone agonist(GnRHa).Methods At 3 weeks of age,42 female Sprague-Dawley rats(brood) were divided into 7 groups(ST dosage groups,as 5 000 ?g/100 g group,200 ?g/100 g group,100 ?g/100 g group,50 ?g/100 g group,25 ?g/100 g group,solvent control group and blank control group)(n=6).Forty-eight female rats were divided into 8 groups(ST therapeutic duration)(n=6).Rats received 2.5 mg/kg im slow-released GnRHa(triptorelin,as 2 d group,3 d group,5 d group,7 d group,10 d group,13 d group,soluent control group and blank control group) which was repeated every 2 weeks for 2 times,3 days after the 2nd GnRHa(D1),ST dosage groups were subcutaneously administrated ST at the various dosage daily(D1-D13).ST therapeutic duration groups were subcutaneously administrated ST at the dosage of 100 ?g/100 g daily for different duration.All the rats were killed on the D14.On the day of sacrifice,body weight,body length and left tibial length were measured,plasma were taken for determining insulin-like growth factor-1(IGF-1),right tibia were fixed,demineralized and processed for paraffin-embedding.Paraff sections were HE stained for growth plate measurements.proliferating cell nuclear antigen(PCNA) on growth plate was analyzed with immunohistochemistry staining and image.Results 1.In the 5 000 ?g/100 g ST dosage group,the weight,Height and tibial length exceeded than those of the other dosage and control groups(Pa