The Study Of Correlation Between Related Cytokine Protein Expression Of Osteoblast In Patients With Osteoporosis And TCM Syndrome Types

Objective:To explore the correlation among related cytokine protein expression of osteoblast in patients with osteoporosis(OP)and "Kidney Yang-deficiency","Kidney Yin-deficiency" TCM Syndrome Types,revelate the essence of osteoporosis TCM Syndrome Types and provide objective base for study about osteoporosis TCM Syndrome Types.Method:The lastest progress of modern TCM's reseach on OP and TCM Syndrome Types of OP was reviewed,the relation of osteoblast(OB)and bone metabolism was summarized,and also reviewed the relation of OP and related cytokine of OB,outlined technology about protein chip and culture of OB in vitro.The study made of part one(experimental study)and part two(clinical study)is as follows. In part one:OB was separated by improved enzymatic digestion from femoral head of three groups patients(Kidney Yang-deficiency group,Kidney Yin-deficiency group,control group)accepted total hip replacement.The morphology of primary osteoblast was observed by inverted microscope,and OB was identified by mineralized nodes with alizarin red staining and the expression of alkaline phosphatase with modified Gomori's assays staining.Positive rate and fluorescence value of Ca²⁺in OB were tested by flow cytometry in three groups, and mineralized nodes were observed and counted.In part two:Three groups patients were all undergone hip replacement, including 8 paients of femoral neck fracture or intertrochanteric fracture with Osteoporosis of Deficiency in Kidney yang,8 paients of femoral neck fracture or intertrochanteric fracture with osteoporosis of Deficiency in Kidney yin and 8 paients of femoral neck fracture or intertrochanteric fracture without osteoporosis.The femoral heads replaced of three groups were chosen to culture OB,the OB cultured showed typical morphy and function through identification.The three groups OB were tested by antibody chip of cytokine in order to select the cytokines whose protein expression was different among Kidney Yang-deficiency group and control group,Kidney Yin-deficiency group and control group,Kidney Yang-deficiency and Kidney Yin-deficiency group.Results:In part one:After enzymatic digestion,OB gained was single and suspended just at the beginning of inoculation,showed spherical shape with high refractive index.2 hours later,OB began to adhere to the wall of training bottles,20 hours later,most of osteoblasts were adherent to the wall of training bottles.About two weeks after culture,OB growed over the wall of training bottles,they were intensive,single long spindle shape.OB growth slowed down at this time to begin a multi-storey growth,and calcium nodules began to appear.12 days after inoculation,alkaline phosphatase staining of OB was operated,it appeared black or massive precipitation in,osteoblasts cytoplasmic granules,widely distributed in the cytoplasm of OB showed high purity.In the existence of ascorbic acid andβ-Glycerophosphate disodium salt pentahy-drate(β-GP), cells cultured for 18 days could formed mineralized nodes,alizarin red stained red.The results showed that the cultured cells in the original showed various features of osteoblasts,ALP testing positive,cells cultured for 14-20 days in the culture medium including ascorbic acid andβ-GP could formed calcified knot which confirmed the cells cultured were osteoblasts with typical osteoblast morphology and function,and high purity.Flow cytometry analysis showed that the positive rate and fluorescence value of OB intracellular Ca²⁺in control group were significantly higher than those in kidney-yang deficiency and kidney-yin deficiency group,there were significant differences(P＜0.01),there was no significant difference(P＞0.05)between kidney-yang deficiency group and kidney-yin deficiency group in the positive rate and fluorescence value of OB intracellular Ca²⁺.It showed that the count of mineralized nodes in control group were significantly higher than those in kidney-yang deficiency and kidney-yin deficiency group,there were significant differences(P＜0.01),there was no significant difference(P＞0.05)between kidney-yang deficiency group and kidney-yin deficiency group in the count of mineralized nodes. In part two:Compared with normal control group,18 cytokines such as BMP-4, BMP-6,FasFGF-4,FasFGF-6 and IGF-1,IL-6,IL-2 protein expression levels of OB in kidney-yang deficiency group and kidney-yin deficiency group were significantly increase or decrease(P＜0.05,P＜0.01),and IL-6,TGF-1 protein expression levels of OB in kidney-yin deficiency group were higher than kidney-yang deficiency group,there were significant differences(P＜0.05, P＜0.01).The essence of OP TCM type may be closely related to cytokines protein expression,we also found that the cytokines which showed difference in protein expression were relevant to bone metabolism,and increase or decrease of the protein expression was closely related to the function of the cytokines.Conclusion:1.Modified enzyme digestion method can gain high-purity OB in keeping with reseach in vitro.2.Various methods can be combined as the identification criteria of osteoblast culture in vitro culture.3.The concentration of OB intracellular Ca²⁺concentration at physiological state is higher than that at osteoporosis state,and there is no significant correlation between calcium intake capacity of OB and "kidney-yang deficiency syndrome","kidney-yin deficiency syndrome".4.The calcification capacity of OB at physiological state is higher than that at osteoporosis state,and there is no significant correlation between calcification capacity of OB and "kidney-yang deficiency syndrome","kidney -yin deficiency syndrome".5.There are similar calcium intake capacity and calcification capacity of OB between "kidney-yang deficiency" OP and "kidney-yin deficiency" OP,the reseach of the OP syndrome type' essence needs discussion at a deeper level.6.The material basis of OP "kidney-yang deficiency syndrome","kidney-yin deficiency syndrome" may be cytokines about bone metabolism.The trend of changes in protein expression of related cytokines of OB in kidney-yin deficiency group were consistent with which in kidney-yang deficiency group.7.The different degree of OB'IL-6,TGF-1 protein expression may be the real differences of OP "kidney-yang deficiency syndrome" and OP "kidney-yin deficiency syndrome".