Objective To investigate the effect and the significance of soft tissue repairing and functional reconstruction in limb salvage operation on extremity malignant bone tumors by individulized prosthesis replacement after malignant tumor resection with the help of Neo-adjuvant chemo-therapy. Methods A total of 78 patients with malignant bone tumor were recruited, including 42 males and 36 females. Aged 19～61, with an average of (29.12±9.47).Tumor in 14 patients was in the proximal humerus, 11 in the proximal femur, 27 in the distal femur, 3 in femoral middle part, and 23 in the proximal tibial. There were 29 cases of osteogenic sarcoma, 18 chondroma sarcomatosum, 7 maligant enchondroma with pathological fracture, 20 maligant giant cell tumor,and 4 maligant inflammatory myofibroblastoma of the bone. Soft tissue repairing and functional reconstruction were carried out together with individualized prosthesis replacement. The type of the prostheses was as follows: 14 patients had long stem humerus head prosthesis, 50 made hinged knee prostheses with femoral or tibial component, 3 whole femur replacements, 7 long stem femoral head prostheses, 4 long stem hip prostheses.Results All patients were followed up for an average of (26.80±8.06) months (4～37 months) and postoperative functions were estimated according to Enneking system. Among the 78 patients, results in 48 (61.5%) were excellent, 17(21.8%) were good,10 (12.8%) were fair,and 3(4.9%) were poor. The satisfactory rate was 83.3%. Conclusion Soft tissue repairing and functional reconstruction in limb salvage operation on extremity bone malignant tumors by individualized prosthesis replacement not only spare the limbs, but also keep their function.It can remove the psychologic obstacles caused by extremity absence, and is effective for bone malignant tumor.

Objective To determine the effect of aldosterone and its antagonist, spironolactone on epithelial-mesenchymal transition (EMT) of normal rat kidney epithelial cells (NRK-52E) in a high glucose milieu,and to explore the mechanism of renoprotection in diabetic nephropathy (DN ) in rats involving aldosterone and spironolacton. Methods NRK-52E cells were simultaneously cultured in the serum-free Dulbecco's modification of Eagle's medium Dulbecco (DMEM) for 12 hours. Then the low glucose (LG) group was cultured in LG (1000 mg/L) DMEM:The high glucose (HG) group was cultured in high glucose (4 500 mg/L) DMEM. The aldosterone (Aldo) groups were cultured in high glucose DMEM with the addition of 10,50 and 100 nmol/L aldosterone respectively. The SP group was cultured in high glucose (4 500 mg/L) DMEM plus 10~(-7)mol/L spironolactone. Immunohistochemistry, RT-PCR and Western blot were used to detect E-cadherin and α smooth muscle actin(α-SMA) mRNA expression. Results RT-PCR showed that compared with the LG Group, E-cadherin mRNA expression in the HG group was significantly lower, and α-SMA mRNA expression was significantly increased(P＜0.05). E-cadherin mRNA expression in the 50 nmol/L Aldo group and 100 nmol/L Aldo group was significantly lower than that in the HG group, while the expression of α-SMA mRNA was significantly increased in the HG group(P＜0.05), with a dose-dependent relationship with aldosterone(r=-0.70,P＜0.05;r=0.67, P＜0.05). E-cadherin mRNA in the SP group was significantly higher,while α-SMA mRNA expression was lower than that in the HG group(P＜0.01). Immunohistochemistry and Western blot showed that compared with the LG group, E-cadherin protein expression was significantly reduced, and α-SMA expression was significantly increased in the HG group(P＜0.01). In the 10 nmol/L Aldo, 50 nmol/L Aldo, and the 100 nmol/L Aldo groups, E-cadherin protein expression was significantly lower, and α-SMA protein expression was significantly higher than that in the HG group(P＜0.05), with a dose-dependent relationship with aldosterone(r=-0.83,P＜0.05;r=0.81, P＜0.05). In the SP group, E-cadherin protein expression was higher, and α-SMA protein level was lower than that in the HG group(P＜0.05). Conclusion Aldosterone can promote EMT of tubular epithelial cells in a high sugar milieu, leading to renal interstitial fibrosis in Diabetic nephropathy. Spironolactone can inhibit high glucose-induced renal tubular epithelial cells EMT, which may be an important mechanism for the inhibition of renal interstitial fibrosis.

Objective To evaluate the efficacy of dural tenting suture and epidural drainage in craniotomy. Methods In 145 cases of intracranial lesions, dural tenting suture and epidural drainage were performed to prevent epidural hematoma. Results Postoperative computed tomography (CT) showed no epidural hematoma required surgery in both groups. Conclusion Both dural tenting suture and epidural drainage are effective in preventing epidural hematoma. Hemostasis is the key step. Dural tenting suture without epidural drainage relieves psychological stress. It decreases the risk of intracranial infection and avoids some unusual complications.

Objective To investigate the efficiency and safety of ultrasound-mediated microbubble destruction in enhancing β-galactosidase gene (β-gal gene) transfer into human proximal tubular cells(HKCs). Methods β-gal gene was transfected to HKCs as a mark gene with ultrasound-mediated microbubble destruction. Cultured HKCs were grouped to receive the following 7 treatments respectively: ultrasound alone; microbubble alone; naked plasmid; ultrasound and plasmid; microbubble and plasmid; ultrasound, microbubble, and plasmid; and VigoFect and plasmid. In Group 6, HKCs were exposed to ultrasound under different sound intensities and time. X-gal stainning, typan blue stainning, and Hochest stainning were used to detect the transfection efficiency, cell survival rate, and cell apoptosis rate, respectively.Results β-galactosidase expression could be observed in the ultrasound-mediated microbubble destruction groups. Along with the increasing of sound intensity and exposure time, the cell survival rate of HKCs decreased, and the cell apoptosis rate increased gradually. The transduction efficiency and survival rate in middle intensity (0.3 W/cm~2×60 s) of ultrasound exposure were higher than those of other groups, similar to those of Group 7.Conclusion Under optimum sound intensity and exposure time, ultrasound-mediated microbubble destruction can increase gene transfer into HKCs. This non-invasive gene transfer method may be a useful tool for clinical gene therapy.

Objective To observe the effect of valsartan on brian ultrastructure, Klotho gene and micro-inflammatory factor [intercellular adhesion molecule-1(ICAM-1) and vascular cell adhesion molecule-l(VCAM-1)] expression in spontaneously hypertensive rat models. Methods Ten male spontaneously hypertensive rats of 22 weeks age were selected and randomly divided into a hypertension group and a valsartan intervention group, while another 5 Wistar-kyoto rats were set as a normal contrast group. The brain ultrastructure of the 2 groups was observed by electron microscope. The expression of micro-inflammatory factor (ICAM-1 and VCAM-1)and Klotho gene was detected with RT-PCR, immunohistochemistry, and Western blot, respectively. Results The cerebral neuron damage of spontaneously hypertensive rats whose ultrastructure showed cell-pyknosis, chromatin margination and typical apoptotic body formation were alleviated after the intervention of valsartan. RT-PCR showed that the gene expression of Klotho increased while ICAM-1 and VCAM-1 decreased after valsartan intervention. Immunohistochemistry and Western blot also showed that the protein expression of Klotho increased, while ICAM-1 and VCAM-1 decreased after valsartan intervention. ConclusionValsartan can improve the brain ultrastructure of spontaneously hypertensive rats by increasing the expression of Klotho.

Objective To determine the aberrant methylation status of RASSF1A,p16 and DAPK gene promoter region in induced sputum from lung cancer patients and the value of their combined detection in diagnosing lung cancers. Methods Methylation-specific PCR (MSP) was used to detect the promoter methylation status of RASSF1A,p16, and DAPK genes in induced sputum and pathological tissues from 82 patients with lung cancers and 25 patients with pulmonary benign lesion.We also analyzed the relation between methylation status and clinical pathological data.Results The positive rates of promoter methylation of RASSF1A,p16, and DAPK genes in pathological tissues from patients with lung cancers were 63.4%,59.8%, and 58.5%, respectively,and those in induced sputum were 54.9%,48.8%,and 51.2%, respectively. The promoter methylation of RASSF1A,p16, and DAPK genes were not detected in patients with pulmonary benign lesion.There was a significant difference between the lung cancer group and pulmonary benign lesion group (P＜0.05). The methylation rate of RASSF1A gene was significantly lower in the middle and high differentiation and non-metastastic lymph node of lung cancer tissues than that in the poor differentiation and the metastatic lymph node of lung cancer tissues(P＜0.05), and was not correlated with age, sex, smoking index, clinical stage, and pathological types.The methylation rate of p16, and DAPK genes was not significantly correlated with all the above mentioned factors (P＞0.05). The methylation rate of joint detecting RASSF1A, p16, and DAPK genes was 73.2%. Conclusion Joint detection for promoter hypermethylation of RASSF1A, p16, and DAPK genes in induced sputum may be used as a simple and effective index of the diagnosis and prognose of lung cancers, and can improve the positive rate.

Objective To investigate the changes of human leukocyte antigen-G (HLA-G) protein expression and Th1/Th2 type cytokines in intrahepatic cholestasis of pregnancy (ICP) and their relativity to the etiology of ICP. Methods Peripheral blood and placental tissues were obtained from 26 ICP patients (the ICP group) and 22 normal pregnant women (the NP group) in the operation room for Cesarean birth. Immunohistochemistry was used to detect the expression of HLA-G protein in the placental tissues. Meanwhile we tested the concentrations of tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) by enzyme-linked immunosorbent assay (ELISA) in the peripheral blood and checked the levels of TBA in the serum.Results TBA level in the ICP group was (27.05±6.08) μmol/L, significant higher than that in the NP group (4.35±2.68)μmol/L (P＜0.01). The positive expression of HLA-G protein in extravillous trophoblast in the ICP group was significantly lower than that in the NP group (P＜0.01). The mean optical density (MOD) of positive expression of HLA-G protein in the placenta tissues in the ICP group (52.91±7.19) was significantly lower than that in the NP group (69.26±7.72) (P＜0.01). The concentration of TNF-α was significantly higher in the ICP group (101.31±19.30) pg/mL than that in the NP group (54.51±23.72) pg/mL (P＜0.01). The concentration of IL-4 was lower in the ICP group (22.16±6.55) pg/mL than that in the NP group (31.69±8.25) pg/mL (P＜0.01). The ratio of TNF-α/IL-4 was higher in the ICP group (4.52±1.91) than that in the NP group (1.72±0.61) (P＜0.01). There was a negative correlation between the MOD of HLA-G protein and TNF-α (r=-0.98, P＜0.01) in the ICP group. No correlation with IL-4 and TNF-α/IL-4 was seen (P＞0.05). There was a positive correlation between TBA and TNF-α (r=0.99, P＜0.01), and a negative correlation between TBA and the MOD of HLA-G protein (r=-1.00, P＜0.01) in the ICP group. No correlation with IL-4 and TNF-α/IL-4 was seen (P＞0.05). Conclusion There is an imbalance of Th1/Th2 cytokines to the Th1 type in the peripheral blood of ICP patients. The expression of HLA-G protein in the placenta of ICP patients decreases, leading to an increase of Th1 type cytokines that may be one of the reasons for liver destroy in ICP.

Objective To analyze the epitopes of anti-hepatitis C virus(HCV)antibodies by peptide library biopanning. Methods Phage random peptide library of 12 amino acids was immunoscreened with purified IgG from the sera of hepatitis C patients. Positive clones which were obtained after 3 rounds of biopanning were detected by ELISA and 4 of them were sequenced. Results After 3 rounds of screening, the radio of output to input increased from (4.6×10~(-4))% to (5.3×10~(-2))%, meaning the enrichment was effective. At the third round of screening, all the selected clones proved to specifically react with the sera for immunoscreening. Four positive phage clones were sequenced, which shared a very conservative sequence and was named as C1. Its inserting sequence in the coat protein III was deduced to be GSMSPYVRWYTP, and the positive rate of C1 reacted with 20 cases of HCV patients was 85%.Conclusion The antigen-mimic peptide C1 is successfully screened from 12 random phage peptide library and it has some diagnostic value.

Objective To construct a prokaryotic plasmid to express the testis development related gene 1 (TDRG1) recombinant protein and obtain anti-TDRG1 mAb by immunizing mice, and to identify the biological properties of the mAb. Methods The coding sequence of TDRG1 was amplified by RT-PCR from normal human testis tissue and cloned into the vector pET21, and then was expressed in the E. coli BL 21(DE3) to get TDRG1 recombinant protein. The purified TDRG1 recombinant protein was injected to immunize the BALB/C mice to develop anti-TDRG1 mAb. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells. The hybridoma cells that secreted anti-TDRG1 mAb were subcloned with limited dilution. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate titers and subtypes of mAb. Western blot and immunohistochemistry were used to detect specificity of mAb.Results The prokaryotic plasmid expressing the recombinant protein was constructed, and the TDRG1 recombinant protein was expressed and purified. Two hybridoma cell lines that secreted anti-TDRG1 mAb were obtained. The titer of the mAb in ascites was 1∶1.6×10~6, and the subtype of the mAb was IgG_1. Westem blot and immunohistochemistry analysis indicated the mAb showed specific combination with TDRG1 protein in human testes.Conclusion The TDRG1 recombinant protein is highly purified and has strong antigenicity. The anti-TDRG1 mAb is produced successfully.

Objective To investigate PAX4 gene polymorphism and its association with islet autoantibody-negative patients with ketosis-prone diabetes in Chinese Han population. Methods We screened the variation of exon 3 and 9 within PAX4 gene by denaturing high performance liquid chromatography(DHPLC) in 112 non-diabetes control subjects (NC group) and 141 patients with ketosis-prone diabetes (KPD group), who were both negative for glutamic acid decarboxylase antibody (GAD-Ab) as well as protein tyrosine phosphatase antibody (IA-2Ab) . The sequences of abnormal peaks were analyzed by DNA-sequencing. The A1168C single nucleotide polymorphism in PAX4 gene was genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 308 non-diabetic control subjects and 141 KPD patients. Results No variation was discovered in PAX4 gene exon 3 both in the patients and in the controls. There was a single nucleotide polymorphism locus A1168C in the PAX4 gene exon 9, which induced mis-sence mutation P321H(rs712701). No significant difference was observed in the genotype and allele frequencies of A1168C polymorphism between KPD patients and control subjects (P=0.532, 0.426). The difference was detected in the CC genotype and C allele frequencies in the KPD group when patients were stratified by gender (P=0.009,0.028). According to age at diagnosis, the difference was observed in the CC genotype and C allele frequencies between <20 years old and ≥20 years old in the KPD group (P=0.034,0.032). The level of FCP in the CC genotype group was significantly higher than that of FCP in AA genotype group (P=0.005). Conclusion A1168C polymorphism in PAX4 gene may not play an essential role in the genetic susceptibility of the islet autoantibody-negative KPD in Chinese Han population. However, A1168C variation may contribute to the predisposition to the male or <20 years old patients with islet autoantibody-negative KPD.