| ObjectiveTo establish model of congenital kidney deficiency in offspring rat and observe its bone development,to verify the "kidney-bone" from t he perspective of congenital kidney essence,and to preliminarily clarif y the relationship between congenital kidney essence and fetal bone de velopment diseases;to culture primary osteoblasts with Zuogui pill con taining ser?m,and to elucidate the possible regulatory mechanism of C x43 protein on the development ofcongenital kidney deficiency in offsp ring.Methods1)Theoretical study: to clarify the relationshi P between kidney and bone and the theoretical mechanism of congenital kidney deficiency aff ecting fetal bone development.2)Thirty pregnant rats(30 SD female rats of SPF class weighing 270±20g and 15 SD male rats weighing 300±20g were prepared in 1:2 cage).The pregnant rats were randomly divided into control group,mo del group and Kidney-tonifying group,with 10 rats in each group.The control group was fed normally.The model group and the kidney-ton ifying group were given simulated earthquake vibration and photoelectri c stimulation combined with stress and intimidation,while the model g roup was given saline and the kidney-tonifying group was given Zuogui pill.All the three groups were fed until the birth of their offspring.Each litter was adjusted to 10 offspring for 5 weeks.The skull suture(1st week),femoral growth plate(3rd week)and femoral cortical bone(5th week)of the offspring were taken and made into sections.HEstai ning was used for morphological observation.Immunofluorescence,RTPCR and Western Blot techniques were used to detect the distribution and expression of Cx43,Sox9,PTHrP.The average length of offspring in 3 weeks,the average length of femur in 5 weeks,the microstructu ral parameters of femoral cortical bone and cancellous bone by microCT,and the contents of calci?m,phosphorus and hydroxyproline in fe mur at 5 weeks were measured to study the development of bone in o ffspring with congenital kidney deficiency.Comparisons were made am ong groups.3)Pregnant rats were prepared in cages of 2,1:2 and divided into c ontrol group and model group.The model group was given simulated earthquake vibration and photoelectric stimulation combined with stress and intimidation,while the control group was fed normal until the of fspring were born.The calvaria of the offspring on the day of birth in the model group were divided into the model group and the kidney-t onifying group.The calvaria of the offspring on the day of birth in th e control group were divided into the model group and the kidney-toni fying group.Osteoblasts from three groups were obtained by enzymatic digestion and cultured in primary osteoblasts.Normal calf ser?m was c ultured in control group and model group.Zuogui pill containing ser?m was cultured in Bushen group and inverted microscope was used to observe the cell morphology.Differentiation and maturation were dete cted: cell growth curve,cell proliferation(MTT),ALP activity(Gomori modified calci?m drilling method),mineralized deposition(alizarin redstaining).The expression of Cx43 in osteoblasts was detected on day 7,14 and 21.The expression of Cx43 in osteoblasts was detected by immunofluorescence staining on day 21,and the function of GJIC was detected by scratch dye labeling tracer.Results1)Pregnant rats were stimulated with combined intimidation during pregnancy,resulting in kidney deficiency and impotence of pregnant rats to nourish fetal rats,resulting in reduced fetal number in the model group(P<0.01 or P<0.05),with significant differences.2)The average body length of congenital kidney-deficiencyrat was from the day of birth to the third week,and it was found that the body length became shorter from the eighth day to the 21 st day(P<0.05),with significant difference.At week 1,3 and 5,the femur length became shorter(P<0.05).3)Morphological observation of the skull suture at week 1.Compared with the control group,the skull suture in the model group was slightly widened,and the number of round and oval osteoblasts and their precursor cells in the suture was decreased.The parietal plate becomes thinner and the barrier space is reduced.4)Morphological observation of the growth plate at the distal end of the femur at week 3 showed that the proliferative layer of the growth plate of the femur in the model group became thinner,and the chondrocytes were disordered and sparse,and there was no significant difference in the morphological arrangement between the chondrocytes in each layer of the growth plate of the bushen group and the control group.Congenital kidney deficiency and insufficiency of kidney essence affect the development retardation of femoral growth plate in the typical part of membranous bone.5)The femur bone mineral salt density was detected at week 5,and it was found that the content of Ca and P in the model group decreased correspondingly to the decrease of bone mineral salt density level,and there were significant differences among the groups(P<0.01 or P<0.05).Femoral masson staining to observe the femoral cortical bone collagen fiber ? type collagen fibers in the matrix,no significant differences between groups in morphology.Micro CT scanning showed that cortical bone thickness,outer diameter circumference and cortical bone area decreased in the model group(P<0.01 or P<0.05).Scanning the microstructure of distal cancellous bone showed that although the thickness,number,bone volume fraction and bone density of the trabecular bone of the model group decreased.6)Results of Cx43 skull suture expressionParaffin embedding slices after immunohistochemical detection Cx43 protein staining for brown in each organization,is the high level expression in the skull bone seam organization,focused on the edge of the joint area of osteoblast and its precursor cells cytoplasm(osteoblast,osteogenesis cells),express lower compared with control group,model group(P<0.05),there is significant difference,compared with model group,kidney expression increased(P<0.05),there is significant difference;WB detection of the content of Cx43 protein in the skull suture: compared with the control group,there was a significant difference in the average gray value of the thinnest bands between the model group and the control group(P<0.01).Compared with the model group,the band thickness and gray scale thickening in the bushen group were significantly different(P<0.05).Rt-pcr detected the expression of Cx43 mRNA in the skull suture: compared with the control group,the content of Cx43 mRNA in the model was significantly reduced(P<0.05).Compared with the model group,the content of Cx43 mRNA in the bushen group was significantly increased(P< 0.05).There was no significant difference between the control group and the kidney-tonifying group.7)Results of Cx43 femur expressionImmunohistochemical detection was performed after paraffin-embedded sections: the femoral growth plate was mainly expressed in the cytoplasm of chondrocytes in the reserve layer and the secondary proliferative layer,and there was no significant difference between the groups(P > 0.05).The femoral shaft cortical bone was expressed in the cytoplasm of osteoblasts at the edge of the trabecular bone and in the bone matrix,and there was no significant difference between the groups(P > 0.05).The content of Cx43 protein in femoral growth plate and cortical bone of femur detected by WB: there was no significant difference in the thickness and gray scale of strips in each group of femoral growth plate(P > 0.05),and no significant difference in the thickness and gray scale of strips in each group of femoral cortical bone(P > 0.05).Rt-pcr detection of Cx43 mRNA content in femoral growth plate and femoral cortical bone: comparison of Cx43 mRNA content in femoral growth plate and femoral cortical bone between groups: Cx43 mRNA expression was observed in all three groups.Compared with the control group,the Cx43 mRNA content of the model decreased,but not significantly(P > 0.05),without statistical significance.Compared with the model group,the content of Cx43 mRNA in the bushen group increased but was not significant(P > 0.05),showing no statistical significance.This experiment adopts the method of immunohistochemical,WB in detection of Cx43 protein,the results show that the femoral growth plates and femoral cortical bone Cx43 protein expression,and the model group were lower,kidney group can improve the Cx43 protein expression,but no statistical difference,the result is consistent with the immunohistochemical results,further confirmed that congenital kidney affect bone development,the main reason is not regulated by Cx43 protein,in the development of the femoral growth plates,immunohisto chemical and WB testing Cx43 protein content results are consistent.Quantitative detection of Cx43 mRNA by Rt-PCR confirmed that all bone tissues of Cx43 were expressed,and the bone growth of congenital kidney-deficiencyrat was slow,but the expression of Cx43 was not significantly correlated with the femur development of congenital kidney-deficiency rat.8)Results of Sox9 growth plate expressionWB detection of Sox9 protein content in femoral growth plate: compared with the control group,there was a significant difference between the model group and the control group(P<0.05).Compared with the model group,the band thickness and gray scale thickening in the bushen group were significantly different(P<0.05).MRNA expression of Sox9 in femoral growth plate detected by rt-pcr: compared with the control group,the content of Sox9 mRNA in the model was significantly reduced(P<0.01).Compared with the model group,the Sox9 mRNA content in the bushen group was significantly increased(P< 0.01).There was no significant difference between the control group and the kidney-tonifying group.As a key regulator of chondrocyte differentiation and chondrogenesis,Sox9 gene can enhance the chondrogenic potential of human mesenchymal stem cells(HMSS).Sox9 gene affects early embryonic development,plays an important role in chondrogenesis,and plays a core regulatory role in early chondrogenic osteogenesis.9)PTHrP expression of femurWB detection of PTHrP protein content in femoral growth plates: compared with the control group,there was a significant difference in the average gray value of the narrowest bands between the model group and the control group(P<0.05).Compared with the model group,the band thickness and gray scale thickening in the bushen group were significantly different(P<0.05).WB detection of PTHrP protein content in femoral cortical bone: compared with the control group,there was a significant difference in the average gray value of the narrowest bands between the model group and the control group(P < 0.05).Compared with the model group,the band thickness and gray scale thickening in the bushen group were significantly different(P < 0.05).Rt-pcr was performed to examine the PTHrP mRNA expression of the femoral growth plate.Compared with the control group,the PTHrP mRNA level of the femoral growth plate in the model group was significantly lower than that in the control group(P<0.05).Compared with the model group,PTHrP mRNA level of femoral growth plate in bushen group was significantly increased(P< 0.05).There was no significant difference in PTHrP mRNA between the bushen group and the control group(P > 0.05).Rt-pcr was performed to detect the PTHrPmRNA expression in the femoral cortical bone.Compared with the control group,the PTHrPmRNA level in the femoral shaft cortical bone of the model group was significantly lower than that of the control group(P< 0.05).PTHrPmRNA levels in femoral shaft cortical bone of bushen group were significantly higher than that of model group(P < 0.05).There was no significant difference in PTHrPmRNA between the bushen group and the control group(P > 0.05).10)PTHrP expression of skull suturesWB detection of PTHrP protein content in the skull suture: compared with the control group,there was a significant difference in the average gray value of the narrowest bands between the model group and the control group(P< 0.05).Compared with the model group,the band thickness and gray scale thickening in the bushen group were significantly different(P < 0.05).Rt-pcr was used to detect the PTHrP mRNA expression in the skull suture.Compared with the control group,the PTHrP mRNA level in the skull suture of the model group was significantly lower than that of the control group(P < 0.05).Compared with the model group,PTHrP mRNA level in the skull suture of the bushen group was significantly increased(P < 0.05).There was no significant difference in PTHrP mRNA between the bushen group and the control group(P > 0.05).The results showed that PTHrP was expressed in the sagittal suture of the skull,and the decreased11)Results of proliferation,differentiation and maturation of primary osteoblasts cultured in skull sutures.MMT method was used to detect the proliferation and cell activity of osteoblasts,and the rising trend was slow in the model group,but there was no significant difference between the groups(P > 0.05).The ALP activity was significantly decreased in the model group compared with the control group(P< 0.05).Compared with the model group,the ALP activity in the bushen group was significantly increased(P< 0.05),with statistically significant difference.Compared with the control group,the area of calcified nodules in the model group was significantly reduced(P<0.05).Compared with the model group,the area of calcified nodules in the bushen group was significantly increased(P < 0.05),showing a statistically significant difference.The results of immunofluorescence detection of Cx43 protein showed that the fluorescence reaction sediments were mainly arranged along the cell membrane surface in the form of linear dot sheets,and the distribution of Cx43 protein in the model group was reduced(P<0.05).Cx43 mrna was detected by rt-pcr,and the results showed that the expression level of Cx43 increased gradually with the progress of cell differentiation.On day 7,the expression level of model group was slightly lower than that of control group and kidney-nourishing group,but there was no statistical significance(P > 0.05).Day 14 and 21 were significantly lower than the other two groups,(P < 0.05).Cx43 protein expression results were corresponding to mRNA expression,and the difference between the control group and the model group was significant(P<0.05).GJIC was detected by fluorescence trace detection method,and the results showed that GJIC function of cells in each group was normal,and the diffusion distance of fluorescence yellow dye between cells was observed.The quantitative fluorescence analysis of dye diffusion area showed that compared with the control group,the quantitative fluorescence of dye diffusion area in the model group was decreased,and there was a significant difference(P<0.01).Compared with the model group,the kidney-tonifying group was significantly increased(P<0.05).ConclusionsThis subject elucidates the theory from the point of view that the deficiency of kidney essence in the mother causes the deficiency of kidney in the offspring to affect their bone development,and puts forward the academic viewpoint that "congenital deficiency of kidney is related to fetal bone disease" for the first time.The method of "kidney essence of pregnantrat with fear of injury" was improved to replicate the animal model of congenital kidney-deficiencyrat.For the first time,Cx43 was used as the entry point to study the mechanism of congenital kidney deficiency affecting bone development in offspring rats,and it was considered that Cx43 mediated GJIC function affecting osteoblast differentiation and maturation was one of the mechanisms of endothelial bone retardation caused by renal insufficiency in offspring rats with congenital kidney deficiency.The conclusions are as follows:1)The pregnantrat were threatened and stimulated by the gestational recombination method,which affected the bone development of the congenital kidney-deficiencyrat in their infancy.Therefore,congenital kidney essence was associated with fetal bone retardation.2)Down-regulated expression of Cx43 is one of the mechanisms affecting the development of cranial sutures in congenital kidney deficiency offspring rats.3)Cx43 mediated GJIC regulation of osteoblast differentiation and maturation is one of the possible molecular mechanisms affecting intracranial suture osteogenesis.4)Renal essence has the same function as Cx43 mediating GJIC,which affects the regulation of bone growth and development by intrathecal bone.Therefore,Cx43 is one of the material bases of renal essence.5)Zuogui pill can treat kidney essence deficiency in pregnancy and prevent bone retardation in children.6)The function of GJIC mediated by Cx43 is one of the mechanisms of zuogui pill for tonifying kidney and filling essence.7)Congenital kidney-deficiency offspring femoral retardation was associated with local PTHrP and Sox9 expression. |
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