| Objective: Diabetic Osteoporosis(OP) is a complication of Diabetes Mellitus on bone. Type 2 Diabetes Mellitus (T2DM) may induce or promote OP by disturbing bone metabolism. T2DM patients have significant high incidence of OP but the mechanism is still not clear. Bone metabolism is a continuous procedure contains old bone renew and new bone formation. And both resorption and formation are involved in the bone turnover. Bone resorption is carried out by osteoclast(OC) while bone formation is by osteoblast(OB). Therefore, plasma biochemical markers involved in bone turnover may correlate with bone turnover rate. Bone-specific alkalne phosphatase(B-ALP) is the enzyme only exists on cell membrane of OB. It may promote bone matrix formation and mineralization when released. In the present study, DM OP model were made by rats with T2DM and ovariectomied. OB was cultured and cell quantity, morphology and grow ability were all measured. Plasma B-ALP level were determined by ELISA. Alterations in both OB and B-ALP level were all checked in normal, osteoporosis, type 2 diabetes Mellitus and type 2 diabetic osteoporosis groups.Method: 100 healthy Wistar female rats were randomly divided into four groups after 1 week feeding: normal control group (NC, N=24), normal ovariectomy group (NOVX, N=26), type 2 diabetes control group (DC,N=24) and type 2 diabetic rats under ovariectomy group (DOVX, N=26). NC and NOVX groups were fed with normal chow diet while DC and DOVX groups were fed with high-sugar-fat diet (which contains 20% sugar, 2.5% cholesterin, 15% cooked lard) for 8 weeks. T2DM were made by intraperitoneal injection of STZ (0.1mmol/L, prepared by citrate sodium, pH4.2, and 30mg/kg) after 12 hours fasting. At the end of 9th weeks, rats with fasting blood glucose(FBG)≥7.8mmol/L and with insulin resistance were considered as T2DM.Rats choosed for NOVX and DOVX group were ovariectomied under anesthesia1 weeks later after T2DM model were done. Bone marrow was collected from rats of NC and DC groups for OB culturing on operation day, which is considered to be the baseline. The whole experiments were taken for 22 weeks. Body weight, fasting blood glucose, INS and B-ALP levels were measured at 0,2,4,6,8,10 and 12 weeks after ovariectoming. Rats were killed at 4,8 and also 12 weeks after ovariectoming and then femur bone marrow were collected for OB culturing. Quantity and morphology of OB were observed by microscope and the 3rd generation of OB was identified by phosphatase staining. MTT method was used for OB grow ability measurement.Data were presented as mean±SDs. Variance analysis or nonparametric tests were used for differences between groups by SPSS 16.0, p<0.05 was considered statistically different.Results:1 FBG levels at 9 weeks: FBG levels in both DC and DOVX Group are significantly higher than those of NC and NOVX Groups,(P<0.01). INS levels in DC and DOVX Group are significantly higher than those of NC and NOVX Groups(P<0.05).2 Serum B-ALP and OB grow ability : there were no differences in serum B-ALP levels between four groups(P>0.05). OB grow ability in NC Group is better than DC Group when observed under inverted phase contrast microscope. MTT rusults: only on the 3rd and 4th day after the 3rd generation OB were cultured, NC Group is better than DC Group(P<0.05).3 FBG, INS and B-ALP levels at 12 weeks(2 weeks): the FBG level in of DOVX Group seems higher than NC Group but didn't reach statistically difference(P>0.05). FBG in DC Group is significantly higher than NC and NOVX Groups(P<0.05),while FBG in DOVX Group is significantly higher than NOVX(P<0.05). FBG has no differences between NC and NOVX groups(P>0.05)while is significantly higher in DC Group when compared with DOVX Group(P<0.05). INS levels in DC Group is significantly higher than the other three Groups(P<0.05). INS in NC Group and DOVX Group are higher than NOVX Group(p<0.05).The serum B-ALP of DC Group is lower than NC Group. There were no differences in serum B-ALP between NC, DC and DOVX Groups. NOVX Group has significantly higher serum B-ALP levels than the other three groups(P<0.05).4 At 14 weeks(4 weeks): there are no differences in FBG levels found levels in all groups. INS in DC and DOVX Group are significantly higher than NC and NOVX Groups(P<0.05). INS in DC Group is higher than the other three Groups(P<0.05), The other had no differences between every two group(p>0.05). The serum B-ALP of NOVX Group is higher than DOVX(P<0.05)while in NOVX is higher than NC Group. DC Group seems have lower B-ALP than NC and DOVX Group but no statistically significance was found(P>0.05). OB from NC and NOVX Group are better than DC and DOVX Group when observed under inverted phase contrast microscope. MTT results showed that : on the 1st,2nd,3rd,5th,6th,7 th of the 3rd generation, OB in NOVX group is better than NC Group(P<0.05), OB grow ability of DC Group is lower than NC Group, while in DOVX Group, it is better than DC Group on 1-7th day culture(P<0.05).5 At 16 weeks(6 weeks): the FBG in DC and DOVX Groups are higher than NC and NOVX Groups(P<0.05), and no differences in FBG levels were found between NC and NOVX Groups, and also between DC and DOVX Groups(P>0.05); There were no differences found in INS among all groups(p>0.05). The serum B-ALP of NOVX Group is higher than NC Group while DC Group is lower than DOVX Group,but there were no significant differences,NOVX Group is significant higher than DOVX Group (P<0.05).6 At 18 weeks(8 weeks): the FBG in DC and DOVX Groups are higher than NC and NOVX Groups(P<0.05).No differences were found in FBG between NC and NOVX Group, and also DC and DOVX Group(P>0.05). It was found that INS in DC and DOVX group had significantly higher than NC and NOVX (p<0.05).The serum B-ALP in NOVX Group is higher than DOVX Group ,but no significant difference was found, DC Group is significantly lower than NC Group and DOVX Group(P<0.05). OB of NOVX Group is better than the other three Groups and DC Group is the worst when observed under inverted phase contrast microscope. MTT rusults showed that: on the 2nd,3rd,4th,5th and 7th day after the 3rd generation of OB were cultured,NOVX Group is better than NC Group, DOVX Group is better than DC Group(P<0.05). On the 1st to 7th day, OB grow ability of DC Group is significantly lower than NC Group(P<0.05).7 At 20 weeks(10 weeks):there were no differences in FBG in four Groups(P>0.05); There were no differences found in INS among all groups(p>0.05).The serum level of B-ALP in NOVX Group and DOVX Group seems higher than NC Group, DC Group seems lower than NC Group and DOVX Group ,but all are not statistically different(P>0.05).8 At 22 weeks(12 weeks):no differences in FBG levels in four groups at this age. There were no differences found in INS among all groups(p>0.05).the serum B-ALP of NOVX Group and DC Group seems higher than NC Group and DOVX Group,DOVX Group seems lower than NC Group,but all don't reach statistical significance(P>0.05); OB from DOVX Group is better than the other three Groups when observed under inverted phase contrast microscope, DC Group is the worst. MTT rusults show that: on the 1st to 7th day after the 3rd genaeration of OB were cultured,NOVX Group is better than NC Group (P<0.05),on the 1st,2nd,3rd,4th,6th and 7th day, the grow ablility of OB from DC Group is significantly lower than NC Group and DOVX Group(P<0.05).Conclusion:1 The growth ability of OB increases after ovariectomied. Serum B-ALP levels increased at the early phrase of disease,and then decreased.2 The growth ability of OB decreased. The serum B-ALP level in rats with of type 2 diabetes mellitus decreased.3 Type 2 diabetes partially offset the increased growth ability of OB and serum B-ALP level in ovariectomized rats.
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