Study Of The Effects Of The Serum Of Kidney-tonifying Traditional Chinese Drugs Fed Rats On Biology Function Of Osteoblasts And The Gene Expression Of Osteopontin

Backgrounds:Osteoporosis is a disease that often happens to old people. Osteoporosis leads the balance of bone formation and bone absorption impaired, makes the loss exceed the formation of bone. Osteopontin (OPN) is a non-collagen protein in bone, which was separated from bone matrix. It has been verified that non-collagen proteins have immediate or subsequent effects on many circulatory and/or cell surface proteins binding to bone. OPN have the same effect for having the same domain -Arg-Gly-Asp (RGD), which is important to form the bone matrix and ore ization of bone. It also has been appraised that kidney-tonifying traditional Chinese drugs can accelerate the fracture union, make the delayed or disunion fracture heal again through no operation. It is used on Primary Osteoporosis patients extensively, but how it effect on the biological function of osteoblasts and the expression of RGD is not clear. Objectives:1. To construct a method of dissociating and cultivating osteoblasts in vitro;2. To investigate the effect of Kidney-tonifying traditional Chinese drugs on the biological functions and the expression of osteopontin of osteoblasts cultivated in vitro;3. To explore the effects of Kidney-tonifying traditional Chinese drugs on the biological functions of osteoblasts and the expression of OPN gene in the osteoblast cultured and osteoblast-osteoclast cocultured system, and to offer the experiment basis for clinical practice of Kidney-tonifying traditional Chinese drugs for treating and preventing osteoporosis.Methods:1. Digging and dissociating osteoblasts from the bones of skull of SD neonate rat by using trypsogen and collagenase. Cultivating osteoblasts in the method of combining cultivating bone flap with cultivating suspension.2. Appraising the cultivated osteoblasts by using the morphology, peculiar enzyme staining and immune histolopathology technology.3. Investigating the mechanism of Kidney-tonifying traditional Chinese drugs anti-osteoporosis in the osteoblasts single cultured and osteoblast-osteoclast cocultured system4. Determining the activity of osteoblasts' ALPase after Kidney-tonifying traditional Chinese drugs on osteoblast and osteoblast-osteoclast cocultured system; prosteoblasying into cell cycle and apoptosis of osteosteoblasylasts by FCM after dealing with Kidney-tonifying traditional Chinese drugs; analysing cell proliferation with the method of MTT and the ablity of forming mineral nodes through staining with ARS after dealing with Kidney-tonifying traditional Chinese drugs in co-cultured system.5. Detect expression of osteopontin and gene osteopontin in every group by Western-blot and by RT-PCR.6. Statistical analytic methods: the software package of SPSS 10.0 is used for the test of significance. Mate t examine and variance analyse are used for measurable materials and LSD method among groups.Results:1. Identify osteoblasts: Osteoblasts take the forms of long shuttleless and triangle type, it sticks wall to grow. It shows large body, clear border, dark red afterbirth, the size and quantity of cell protruding vary, its nuclear locates in the edge of thecell, takes the form of round or oval, 1-2 kernels in cell is blue; the positive stain of type I collagen immune histolopathology and ALPase is the characteristic qualities of osteoblasts, Thus the cultured cell can be appraised to be osteoblasts. 2. (1) The activity of ALPase changes from(0.768 ± 0.046)(hign dozes group , HD), (0.787+0.041) (middle dozes group ,MD), (0. 780+0.028) (Estrol group, E2), (0.559 ±0.070) (Control group ), to(0.598± 0.045) (low, LD). The activity of ALPase increase notably in MD, HD and E2 groups compared with control group (P <0.01), and in E2 group also arises significantly compared with in MD, HD groups(P<0.05).(2) The cell cycle changs as the percentage of( G 2+ S )period from (10.24+ 1.85) %(HD)> (12.10 + 1.42) % (MD), (11.10+1.21) %(E2), (7.57+1.05) % (Control), (7.69 +1.77) % (LD) .The percentage of( G 2+ S )period in MD, HD and E2 groups increase notably compared with in control group ( P <0.01), and in E2 group also arises significantly compared with in MD, HD groups( P <0.05). It indicates that synthesis of cell DNA and cell division increase.(3) The osteoblasts proliferate largely and at the same time the apoptosis decrease .The value of osteoblasts changes in different groups [(0.695 + 0.022)(HD)^ (0.714 + 0.024) (MD)> (0.758 + 0.037) (E2)> (0.492 + 0.036) (Control)]. The value of osteoblasts in MD, HD and E2 groups increase higher than in control group ( P <0.01), and in E2 group also arises significantly compared with in MD, HD groups(P <0.05);(4) The apoptosis varied from (22.57+5.45) % (HD), (20.55 ±1.39) % (MD), (20.43±2.37) % (E2), (28.50±2.84) % (LD), to (30.63 + 3.15) % (Control).MD, The apoptosis in MD,HD and E2 groups decreased notably compared with in control group (P <0.01).(5) The mineral nodes change from (286.00±7.38) (HD)> (293.56± 8.92) (MD) (301.33±10.51) (E2), (248.44± 11.50) (Control) to (253.67±14.18)(LD). Themineral nodes in MD,HD and E2 groups increase obviously notably compared within control group (P <0.01). (6)The rate of OPN / ^ -actin in each group are (0.670 ±0.042)(HD), (0.700+0.015) (MD), (0.680+0.040) (E2), (0.546+0.040) (Control) and (0.578+0.044) (LD). The gene expression of OPN in HD, MD, E2 groups increase signifiangtly compared with in control group (p<0.01). The result of semiquantitative RT-PCR displayed that the serum of Kidney-tonifying traditional Chinese drug-fed rats could increase the gene expression of OPN in the single culture system;3. After being dealed with the the serum of Kidney-tonifying traditional Chinese drug-fed rats in co-culture system, the activity of ALPase, mineral nodes and gene expression of OPN have changed.(1) The activity of ALPase in MD, HD and E2 groups increase higher than in control group ( P <0.01), and in E2 group arises significantly compared with in MD, HD groups( P <0.05);They are relatively higher than in the single culture system in corresponding groups.(2) The result of semiquantitative RT-PCR displayed that the serum of kidney-tonifying traditional Chinese drug-fed rats could regulate up the expression of RGD gene in the co- culture system. Corresponding, mineral nodes in Kidney-tonifying traditional Chinese drugs in MD and HD groups, E group are higher than contrast group, but they still relatively lower than in the single culture system (P<0.05). Conclusions:l.Osteoblasts cultivated successfully by the way of combining cultivating bone flap with cultivating suspension digged and dissociated from the bones of skull of SD neonate rat by using trypsogen and collagenase 1.2. Kidney-tonifying traditional Chinese drugs can promote the function of osteoblasts' bone formation.3. Kidney-tonifying traditional Chinese drugs can promote the function of bone formation. Mineral nodes increasing maybe be due to the gene expression of OPN up-regulated.4. It is distinct that the effects of the Kidney-tonifying traditional Chinese drugs on forming the mineral nodes in different cultured systems, which may be relative with the level of expression of OPN.