Objective To explore the feasibility of mouse muscle-derived cells with adenovirus-mediated expression of BMP-2 in the treatment of mouse tibia bone defect via collagen sponge as scaffold. Methods Mouse adenovirus BMP-2 plasmids were prepared and identified by PCR.The adherent cells of muscle-derived stem cells cultured in vitro were passaged,cells of the third generation were selected,and recombinant Ad-BMP-2 plasmids were expressed and incubated in mouse myoblasts.The number of BMP-2 positive cells was measured using indirect immunofluorescence method,and alkaline phosphatase(ALP) method was adopted to determine the cell activity in each group.The mouse models of proximal tibial bone defect were established(n=80) and were divided into Ad-BMP-2 transfection group,Ad-LacZ transfection group,blank group and Ad-BMP-2 implantation group.The histological and radiological changes were observed 3 weeks after operation. Results It was revealed by indirect immunofluorescence that BMP-2 positive cells were frequently found in Ad-BMP-2 transfection group,while none were observed in Ad-LacZ transfection group and blank group.The ALP activity was higher in Ad-BMP-2 transfection group,while that was extremely low in Ad-LacZ transfection group and blank group.There was most typical imaging of repair and healing of bone defect in Ad-BMP-2 transfection group,followed by Ad-BMP-2 implantation group,while there was no typical imaging in Ad-LacZ transfection group and blank group. Conclusion The constructed Ad-BMP-2 plasmids can effectively transfect mouse muscle-derived cells,and the transfected cells can continuously express effective amount of BMP-2.The mouse muscle-derived cells with adenovirus-mediated expression of BMP-2 play a definite role in the treatment of bone defect,which provides an effective method and material for clinical application.

BACKGROUND: With the improvement of biomaterial and further recognition in aetabular revision, the main tendency is using biologic prosthesis for mild or moderate acetabular bone defect in total hip revision. OBJECTIVE: To observe the clinical effect of simple particulate bone grafting on mild or moderate acetabular bone defect in total hip revision. DESIGN, TIME AND SETTING: A retrospective case analysis. All cases were from Department of Orthopedics, Shanghai East Hospital Affiliated to Tongji University from June 2003 to June 2008. PARTICIPANTS: A total of 10 patients with mild or moderate acetabular bone defect who received total hip revision were from Department of Orthopedics, Shanghai East Hospital Affiliated to Tongji University. Among them, there were 5 male and 5 female, with an average of 58.8 years old from 43 to 77 years old. Their last replace time were 3-10 years, with an average of (7.0?0.5) years. Two of them in the last replacement used cement components, three used cementless, four used mixture, and one used dipolar artificial thigh bone. According to AAOS typing, there were 6 cases of type Ⅰ and 4 cases of type Ⅱ. The average preoperative Harris hip joint score was 40.6. METHODS: Simple particulate bone grafting combined with biologic prosthesis was used to treat the 10 patients with mild or moderate acetabular defects. All operations were at the posterolateral approach. The acetabular cup and screw fixation were performed after grafting. The postoperative effect should be evaluated by clinical and X-ray manifestations. MAIN OBJECTIVE INDEX: The bone grafting time, bleeding volume, Harris score and complications in the perioperative period were recorded. RESULTS: All patients were adopted simple particulate bone grafting combined with biologic prosthesis to reconstruct the acetabular bone defects. One acetalum cup was found loose after one month. The titanium plate combined with particulate bone grafting was used in the second revision. At 3 months after operation, the patient could walk without crutch. No complication was found in other patients. An average of 3 years following-up was obtained. The average Harris score was 87.0. Except one acetalum cup was found loose, the X-ray showed that the bed of acetabulum and particulate bone was good healing and there was no complete transparent zone. There was no patient with displacement of acetalum cup. No osteolysis in any patient. The particulate bone and host bone had a good healing. CONCLUSION: The simple particulate bone grafting is simple and effective for mild or moderate acetabular bone defects with the good follow-up result in early period.

Objective To investigate the effects of MG132 on diabetic nephropathy (DN) rats induced with streptozocin. Methods Seventy-two male SD rats were randomly divided into three groups: normal control group (NC, n=24), DN group (n=24) and DN treated with MG132 group (DN+MG132, n=24). At the end of 4, 8 and 12 weeks, 24 hour urinary protein excretion rate (UPER) was detected. Morphology of kidney was examined by special staining of periodic acid-schiff (PAS). Renal 26S proteasome activity was determined by quantifying the hydrolysis of S-LLVY-AMC in a fluorescence reader. Urinary malondialdehyde (MDA) level and renal SOD and GSH-PX activity were detected by commercial kits. Renal SOD, GSH-PX and p47phox mRNA expressions were determined by real-time fluorescence PCR. Renal p47phox protein expression wasdetermined by Western blotting. Results Compared with NC group, the DN group showed a significant increased of UPER at week 4, 8, 12 (all P<0.05), of mesangium proliferation and mesangial matrix expansion at week 12. In DN+MG132 group, UPER was significantly decreased compared with DN group at the end of 4, 8 and 12 weeks (P<0.05, respectively), and the glomeruler pathological alteration induced by diabetes was attenuated. Increased renal 26S proteasome activity in DN rats was significantly inhibited after MC132 administration (P<0.05). Moreover, renal p47phox mRNA expression in DN group was 155%, 149% and 120% more than those in NC group at 3 time points (all P<0.05), and so was the renal p47phox protein expression, 139%, 152% and 186% more (all P<0.05). Urinary MDA levels in DN group were 1.95-, 2.04-and 2.62-folds more than those in NC group (all P<0.05). In addition, compared with NC group at 3 time points, in DN group, renal SOD activity was decreased by 23.09%, 33.59% and 53.31% (all P<0.05); renal GSH-PX activity was decreased by 28.57%, 33.06% and 48.76% (all P< 0.05); renal SOD mRNA was decreased by 38.09%, 61.44% and 76.53% (all P<0.05); renal GSH-PX mRNA group was decreased by 29.16%, 37.26% and 62.40% (all P<0.05). Compared with DN group, renal p47phox mRNA and protein expression, and urinary MDA levels were significantly lower in DN+MG132 group (all P<0.05); renal SOD and GSH-PX activity as well as mRNA expression were significantly increased in DN+MG132 group (all P<0.05). Conclusions MG132 treatment can provide renoprotection for DN rats effectively maybe through enhancing renal anti-oxidative ability.

Objective To investigate the effect of 4-phenylbutyric acid(4-PBA)on the renal pathogenesis of rats with streptozotocin-induced diabetes and its mechanism. Methods Fifty-four male SD rats were randomly divided into three groups:normal control group(NC group,n=18),diabetic nephropathy group(DN group,n=18),diabetic nephropathy plus 4-PBA treatment group(4-PBA group,n=18).At the end of 4,8 and 12 weeks,index of kidney weight/body weight ratio(KI)were measured and calculated.Serum creatinine (Scr),blood urea nitrogen(BUN),urinary MDA levels,urinary SOD activity,and 24 hour urinary protein excretion ram(UAER)were detected by HITACHI automatically.Morphology of kidney wag examined by special staining of periodic acid-schitt (PAS).The p47phox and nitrotyrosine (NT) expression in kidney were determined by real-time fluorescence PCR and Western blotting. Results Compared with the NC group, the DN group rats showed a significant increase of KI(P＜0.05), UAER(mg/24 h) (4.92±0.70 vs 0.26±0.07, 5.29±0.83 vs 0.28±0.08, 5.54±0.81 vs 0.29±0.04,respectively, P＜0.05]for indicated time, mesangial cells proliferation and mesangial matrix expansion at 12 week. However,4-PBA treatment could significantly inhibit the increase of KI (P＜0.05), decrease UAER (mg/24 h) (3.71±0.37, 3.47±0.36, 3.28±0.40, respectively, P＜0.05]for indicated time, and prevent the glomeruler pathological alteration induced by diabetes. Moreover, the mRNA expression of p47phox in the kidney of DN group was 154.72%, 148.60% and 91.95% more than that of NC group (all P＜0.05) for indicated time. The protein expression of p47phox was 118.00%, 140.10% and 177.82% more than that of NC group (all P＜0.05), and the protein expression of NT was 45.29%,59.13% and 89.28% more than that of NC group (all P＜0.05). In addition, urinary MDA levels in DN group were 2.05-, 2.26- and 2.43- folds of NC group, and urinary SOD activities were decreased by 64.78%, 71.29% and 79.32% of NC group. Compared with the DN group, the mRNA and protein expression of p47phox, and protein expression of NT in 4-PBA group were decreased markedly (all P＜0.05) at the end of 8 and 12 weeks. The urinary MDA level was decreased, and the urinary SOD activity was increased significantly in rats with diabetes after 4-PBA treatment for indicated time (all P＜0.05). Conclusion 4-PBA treatment can significantly inhibit the renal pathogenesis of rats with diabetes through inhibition of oxidative stress.

Background Researches showed that multifocal electroretinogram (mfERG) is able to assess the retinal function in the eyes with acute Vogt-Koyanagi-Harada ( VKH ) syndrome. But the mfERG characteristics of convalescence stage of VKH are still below clear. Objective Present study was to compare and follow up the variation process of visual acuity and mfERG in acute and recovery stages of VKH syndrome. Methods This was a clinic-based retrospective study. Visual acuity, mfERG and fundus fluorescence angiography ( FFA ) were recorded from 35 eyes of 18 acute VKH cases. The period of follow-up in recovery stage lasted about 18 months with the repetitive recording results for 4 times. Results In this study, the visual acuity range in acute stage VKH was 0. 01 to 1.0, and 91.4% (32/35 eyes) was below 0.6. Compared with normal control group, the visual acuity was significantly decreased (P＜0.01). The response densities (amplitudes) of N1 ,P1 waves of the first-order kernel were significantly lowed in all the 6 rings,and the implicit times of 1-4 rings of both waves were significantly prolonged in acute VKH eyes(P＜0. 05). The abnormalities of retinal function showed a regional difference at the posterior pole retina with the dominant change in the first ring,showing a cutting off78% in the P1 amplitude. The abnormal degree of mfERG was more serious as the the increase of retinal eccentricity. In 2 months of convalescence after glucocorticosteroids therapy,the range of visual acuity were 0. 1-1.2 ,and the amplitudes of N1, P1 of 1-2 rings were greatly elevated in comparison with acute on-set (P＜0. 05 ). However, there was still a remarkable difference in the amplitudes of from 1 through 6 rings,comparing with normal. The response density of P1 wave from whole recording region was only 44% of normal. Though the visual acuity was stable during the follow-up duration, a decreasing tendency in N1 and P1 amplitudes were seen. The implicit times of both wave shortened only in 1-3 rings in recovery stages of VKH (P＜0.05). Conclusion VKH syndrome cause serious damage of posterior retinal function.Macular region is the site with greater retinal functional lesion and restore before and after medication. This hardly recovery of retinal function can last over one and half year,even satisfied visual acuity is stable after proper treatment.

Background The pathological change of the anterior uveitis is the disruption of blood-aqueous barrier.Slit lamp examination appears to be limited for the evaluation of inflammatory response,and fluorescine angiography is an objective approach.However,there are few relative studies up to now in China.Objective Aim of this study was to observe the characteristics and assess the clinical applications of iris fluorescein angiography (IFA)in Chinese uveitis with brown iris.Methods Forty eyes of 40 normal subjects and 21 eyes of 13 patients with the anterior uveitis were collected in this study.IFA,slit-lamp examination and iris photograph were performed on the subjects.All individuals were informed consented at the initiation of this study.Results In normal eyes,fluorescence in iris vessels was blocked by the melanin pigment,but peripupillary weak fluorescent leakage was seen in the normal eyes with the age of ＞60 years old.The multiple patterns of fluorescence leakage were found in the patients suffered from uveitis of various etiologies although the negative slit-lamp finding,including the leakage of fluorescein around the pupillary margin and radial iris vessels in the eyes with mild diseases,transmitted fluorescence of regular iris vessels in the eyes with diffuse and local iris atrophy,and vascular tufts of the pupillary margin with coiled interwind tight clusters of thin vessels at the early phase in the eyes with dilated capillaries,microvascular anomalies and new vessel formation.The hyperfluorescence remained throughout the IFA duration.Conclusions IFA findings in uveitis vary depending on the topography,type and severity of inflammation.IFA has a good clinical applying value because of its objective assessment ability of the degree of the blood-aqueous barrier breakdown and iris neovascularization breakdown.It can exhibit the unvisible lesion under the slit-lamp and monitor the efficacy of medical theraphy in patients with active or quiescent uveitis.

Objective To study the role of endoplasmic reticulum stress in phenotypic change of cultured human glomerular mesangial cells induced by high glucose.Methods Cultured human glomeruar mesangial cells were divided into three groups: control group,high glucose group and high glucose+ 4-phenylbutyric acid (4-PBA) group.Cell number of proliferation was assessed by MTT assay.Cell cycle was measured by flow cytometric analysis.Expression of α-SMA was assessed by immunohistochemistry and was observed by laser scanning confocal microscope.Involved mRNA and protein expression were measured by real-time PCR and Western blotting.Results (1)Cell number of proliferation and S transition proportion in high glucose group significantly increased than that in control group (P ＜ 0.05).High glucose could induce α-SMA expression significantly (P＜0.05).4-PBA could significantly inhibit human glomerular mesangial cells proliferation (P＜0.05),S transition arrest (P＜0.05) and expression of α-SMA (P＜0.05) induced by high glucose.(2) Compared with control group,high glucose could significantly increase the expression of glucose-regulated protein78(Grp78 ) mRNA and protein (P＜ 0.05),which could be inhibited by 4-PBA treatment (P＜0.05).(3)High glucose could induce the mRNA and protein expression of TGF-β1 and FN significantly,which could be inhibited by 4-PBA treatment (P＜0.05).Conclusion Endoplasmic reticulum stress plays an important role in phenotypic change of cultured human glomerular mesangial cells induced by high glucose.

In order to establish the stable andreliable ISSR-PCR System of Lysimachia christinae, L16 (4(5)) orthogonal design, which based on 7 levels of single factor experiment, were used in this study. The variance analysis was carried out by SPSS 19.0, and 5 main factors affecting the reaction system were optimized in 4 levels. The best annealing temperature was selected by the optimized reaction system. And the stability and reliability of this system was tested by 23 samples from different origins. The results showed that the five factors (DNA template, primer, dNTP, Mg2+ and Taq enzyme) were the most impacts on the amplified results of ISSR-PCR of L. christinae. The order of the influence was: primer > Taq enzyme > DNA template > Mg2+ > dNTP. The optimal system, which was determined by multiple comparison on different levels of each factor, was total volume of 25 microL, including DNA template 60 ng, primer 0.3 micromol x L(-1), dNTP 0.2 mmol x L(-1), Mg2+ 1.8 mmol x L(-1), Taq enzyme 1.25 U. The optimal system was stable and reliable tested by 23 samples from different origins. This study lays the foundation for genetic diversity analysis, fine varieties selection and molecular identification of L. christinae, and provides reference for optimization on ISSR-PCR system of other speciesin future.
DNA Primers ; genetics ; DNA, Plant ; genetics ; Drugs, Chinese Herbal ; chemistry ; classification ; Microsatellite Repeats ; Polymerase Chain Reaction ; methods ; Primulaceae ; classification ; genetics ; Quality Control

Objective To investigate the correlation between plasma proteasome and endothelial dysfunction in patients with uremia. Methods Forty-five uremic patients who did not receive hemodialysis were defined as A group; seventy-five uremic patients who had received hemodialysis for 6 to 12 months were divided into sufficient hemodialysis group (44 cases,B group)and insufficient hemodialysis group (31 cases,C group).The primary disease of these patients was chronic glomerulonephritis.Fifteen healthy people were defined as healthy control group (D group).The diameter of radial artery lumen (DRL),intima-media thickness (IMT),intima-media area (IMA),endothelium-dependent or independent dilation (EDD or EID) of radial artery in right forearm were detected by diasonography.The levels of 20S proteasome,tumor necrosis factor α (TNF-α),C-reaction protein (CRP) and transforming growth factor β 1 (TGF-β1) of plasma and supernatant of cultured human umbilical veins endothelium (HUVEC) were determined by enzyme linked immunosorbent assay (ELISA).20S proteasome activity was analyzed by special substrate.Results Compared with D group,the level and activity of 20S proteasome,as well as TNF-α,CRP and TGF-β1 in A,B and C groups were significantly increased.Compared with A group,these plasma indices levels were significantly decreased in B group but strongly increased in C group.IMT and IMA were elevated,while DRL,EDD and EID were decreased significantly in A,B and C groups when compared with D group.These parameters were worse in C group than those in A and B groups.After co-culture of HUVEC with above mentioned human uremic serum,the level and activity of 20S proteasome and TNF-α were higher in A,B,C groups than that in D group.In A and C groups,there were negative correlations of EDD with the level or activity of 20S proteasome,TNF-α,CRP and TGF-β1,and there were positive correlations of 20S proteasome level or activity with TNF-α,CRP and TGF-β1. Conclusions 20S proteasome level and activity are significantly increased in uremic patients.There is a close correlation between 20S proteasome and endothelial dysfunction of radial artery.

OBJECTIVETo analyze the short-term and long-term outcomes of the three operation methods in treatment of secondary hyperparathyroidism.

METHODSClinical data of 88 patients who underwent parathyroidectomy for secondary hyperparathyroidism from October 2004 to October 2014 were reviewed retrospectively. Patients were divided into three subgroups, including subtotal parathyroidectomy (group I), total parathyroidectomy with autotransplantation (group II) and intraoperative ⁹⁹Tc(m)-MIBI radioguided total parathyroidectomy with autotransplantation (group III). Their serum calcium, phosphorus, serum iPTH results in the pre-operation, short-term (≤ 6 months) and long term (> 6 months) of post-operation were collected and compared. The improvements of clinical symptoms together with the postoperative recurrence rate and the complication data were observed and analyzed.

RESULTSThe symptoms of all patients were improved after the operation.The short-term postoperative serum calcium, serum iPTH and recurrence rate of Group I and group II were all not statistically significant. The short-term postoperative serum calcium, iPTH and recurrence rate of group III were significantly lower than those of group I and group II (P < 0.05). Postoperative serum phosphorus values of the three groups had no statistical difference. The operation time of group III was significantly shorter than that of group II ((77 ± 13) vs (108 ± 17) min, P < 0.05). The positive rates were more accurate in group III than in group II (98.5% vs 88.7%, P < 0.05). For the long term follow-up, the postoperative serum calcium, phosphorus, serum iPTH and recurrence rate of group I and group II were not statistically different. The postoperative recurrence rate of group III was lower than that of group I and group II (3.6% vs 31.6% and 21.4% respectively, P < 0.05).

CONCLUSIONSTotal parathyroidectomy with autotransplantation has a good efficacy to treat secondary hyperparathyroidism. The intraoperative ⁹⁹Tc(m)-MIBI radioguided total parathyroidectomy with autotransplantation can find the ectopic and supernumerary glands to improve the success rate of operation. And it also can permit omission of frozen section to reduce the operative time.In the postoperative follow-up, the recurrence rate is low, so it is a safe and effective treatment.

Calcium ; blood ; Humans ; Hyperparathyroidism, Secondary ; surgery ; Parathyroid Glands ; surgery ; Parathyroid Hormone ; blood ; Parathyroidectomy ; methods ; Phosphorus ; blood ; Postoperative Period ; Recurrence ; Retrospective Studies ; Transplantation, Autologous ; Treatment Outcome