Objective To construct pcDNA 3.1(+)/HPV 18 E 6 fusion gene and a single-codon mutation pcDNA 3.1(+)/E6 F49R or pcDNA 3.1(+)/E6 F127R fusion gene in eukaryotic expression vector and study the effects on proliferation and apoptosis of cervical carcinoma cell line Hela. Method HPV 18 E6 gene sequence and the single-point mutation HPV 18 E6 F49R or HPV 18 E6 F127R were amplified from total RNA of Hela cell line by reverse transcription- polymerase chain reaction ( RT- PCR), then the gene sequences were respectively inserted into pcDNA 3.1(+) vector to reconstruct recombinant plasmids which were transfected transiently into Hela cells. MTT and RT-PCR were used to test the expression levels of HPV 18 E6 and the growth of HeLa cells after transfected about 48 h. The proliferation and apoptosis of Hela cells were detected respectively by cell counting and AO/EB fluorescent vital staining. Results The pcDNA 3.1(+)/HPV 18 E6, pcDNA 3.1(+)/E6 F49R and pcDNA 3.1(+)/E6 F127R eukaryotic expression vectors were successfully constructed. The gene of HPV 18 E6 was discriminably detected in the HeLa cells which were transfected with the recombinant plasmids. After several days, the proliferation of Hela cells transfected with pcDNA 3.1(+)/E6 F49R or pcDNA 3.1(+)/E6 F127R plasmid were obviously inhibited and the apoptotic rates were significantly increased, then the proliferation of cells transfected with pcDNA(+)/HPV 18 E6 was rather increased slightly, and we could observe the phenomena of early apoptosis and the formation of thekaryopyknosis by fluorescent microscope in the cells transfected with pcDNA 3.1(+)/E6 F49R or pcDNA 3.1(+)/E6 F127R. Conclusion The eukaryotic expressing vectors encoding HPV 18 E6 F49R and HPV 18 E6 F127R provide fundamental basis for the further study on HPV 18 E6 mechanism as well as prevention and treatment of uterine cancer.

Objective To synthesize 4 kinds of 111 In?TPP cations and evaluate their properties as tumor cationic radiotracers in vivo and in vitro. Methods DO3A?xy?TPP, DO3A?xy?mTPP, DO3A?xy?dmTPP and DO3A?xy?tmTPP were radiolabeled with 111 In;their lipid?water partition coefficients and in vivo and in vitro stability were evaluated. The binding affinities of 4 kinds of 111 In?radiotracers were determined in cell uptake and cell efflux assay using U87MG tumor cells. Biodistribution studies and γ imaging studies were performed using the athymic nude mice bearing U87MG human glioma xenografts to explore the biologi?cal properties of 4 kinds of 111 In?radiotracers. One?way analysis of variance was used. Results The labeling yields of 4 kinds of 111 In?radiotracers were all above 85%, and the radiochemical purity were all greater than 99% after purification. Binding assay in U87MG cells showed that 4 kinds of radiotracers had great binding affinity and cell retention ability, and 111In?DO3A?xy?mTPP had the best binding ratio (1?49%;F=177.8, P<0.05) . Gamma imaging and biodistribution results showed that the U87MG tumors could be clearly visualized by 111In?DO3A?xy?mTPP, 111In?DO3A?xy?dmTPP and 111In?DO3A?xy?tmTPP, and the liver uptake of the 3 tracers was lower than that of 111In?DO3A?xy?TPP. In particular, 111In?DO3A?xy?mTPP had the best tumor/liver ratio (0.13±0.05, 2 h postinjection;F=9.4, P<0?05). Conclusions The tumor?targeted ability of 111In?DO3A?xy?mTPP is better than those of 111In?DO3A?xy?dmTPP, 111In?DO3A?xy?tmTPP and 111In?DO3A?xy?TPP, suggesting that it has the potential to be a promising tumor cationic radiotracer.

Objective To compare the effect of 89Sr radionuclide therapy and radiotherapy in the treatment of metastatic cancer pain of vertebral column. Methods 80 patients with cancer pain of vertebral metastasis in the second People's hospital of Yibin from April 2015 to April 2017, were randomly divided into the control group treated by radiotherapy treatment (n=40) and the the observation group treated by 89Sr radionuclide therapy(n=40), and the effect of treatment were compared between two groups. Results In the observation group, the total effective rate of pain response in patients with metastatic cancer pain of vertebral column was 85.0％, and the control group was 82.5％. There was no significant difference between two groups. The onset time of treatment in the observation group was (6.5±1.7)d, significantly shorter than that of the control group (12.9±2.6)d, and the difference was statistically significance (P<0.05). Conclusion The application of 89Sr radionuclide therapy in the treatment of vertebral metastatic pain is equivalent to the radiotherapy in improving the pain response, but the efficacy could be achieved in a short period of time, so it is worth popularizing widely in clinical practice.

BACKGROUND: Many operations for isolating, purifying and identifying bone marrow mesenchymal stem calls (BMSCs) are complicated and cost much. Also they have great effect on cell activity. Whether whole bone marrow adherent culture can avoid above-mentioned disadvantages remains unclear. At present, many studies huve been done to confirm an effective and low cost method for isolating, purifying and identifying such cells.OBJECTIVE: This study is to in vitro induce and differentiate rat BMSCs by whole bone marrow adherent culture,and to identify the cells.DESIGN: A controlled observational experiment.SETTING: Qingdao University Medical College.MATERIALS: This study was carried out in the Laboratory of Oral Cavity and Laboratory of Molecular Biology (provincial level) Qingdao University Medical College between November 2005 and March 2007. Twenty Wistar rats of either gender, aged 3 to 4 weeks, of SPF grade, weighing 120-150 g, were provided by the Qingdao Laboratory Center. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals. Fetal bovine serum (FBS, Hangzhou Sijiqing Bioengineering Material Research Institute), alkaline phosphatase (ALP) kit (Nanjing Jiancheng Bioengineering Research Institute), reverse transcription kit (American Promega Corporation) and primer (Shanghai Bioengineering Co.,Ltd.) were used in this study.METHODS: Adult rat BMSCs were isolated and cultured by whole bone marrow adherent culture. They were digested with 2.5 g/L trypse and inoculated at a density of 5 ×107 L-1 in 6-well culture plate. Then, the cells were divided into experimental group and control group. Inducing culture medium was added to experimental group, and the same amount of basic culture medium was added to control group. ① Cell differentiation and calcium tuberculation were observed under the inverted microscope. ② Biological characteristics of induced cells were detected by calcium tubercle Von Kossa and alizarin Bordeaux. ③ALP activity was detected by diazo salt staining. ④Human core binding factor alpha subunit-1 (Cbf α-1), osteocalcin (OCN) and osteoblast-specific Osterix (OSX) mRNA expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR).MAIN OUTCOME MEASURES: ① Induction and differentiation results of cells. ② Biological characteristics of cells induced by rat BMSCs. ③ ALP activity. ④ Cbf α-1, OCN and OSX expressions.RESULTS: ①Inducing culture medium was added in the serial subcultivation. About 9 days later, cell clones were connected to each other. On about 21 to 28 days, some pykno-round mineralized tubercles appeared. Meanwhile,control cells were connected to each other, but they did not form the tubercle. ② In the experimental group, when MSCs were induced for 21 to 28 days, obvious round or oval calcified tubercles were seen by naked eyes. The results of Von Kossa staining exhibited black sediments, and those of alizarin Bordeaux staining exhibited salmon tubercles. Calcium tubercles were not found in the control group. ③The ALP activity after 2 weeks of induction was obviously increased in the experimental group, but was relatively weak in the control group. ④In the experimental group,Cbf α-1, OCN and OSX expressions were significantly increased after induction.CONCLUSION: After being in vitro induced and differentiated by whole bone marrow adherent culture, rat BMSCs exhibited morphological and biological characteristics similar to typical osteoblasts.

Objective To investigate the changes in serum matrix metalloproteinase(MMP)-2 and MMP-9 and their relationship with serum B-type brain natriuretic peptide(BNP)in patients with chronic heart failure.Methods MMP-2,MMP-9 and serum BNP levels were measured in 184 patients with chronic heart failure and 61 healthy controls.The relationship between changes in MMP-2 and-9 and serum BNP was analyzed.Results Chronic heart failure was categorized into grade Ⅱ,Ⅲ and Ⅳ according to NYHA.In grade Ⅱ,Ⅲ and Ⅳ and the control group,the levels of MMP-2 were(309.1±60.1)nmol/L,(422.6±89.6)nmol/L,(694.8±126.2)nmol/L and(217.2±26.3)nmol/L respectively,and the levels of MMP-9 were (321.2±63.2)nmol/L,(454.4±96.3)nmol/L,(634.1±51.2)nmol/L and(210.8±23.6)nmol/L respectively.The levels of MMP-2 and MMP-9 were significantly higher in chronic heart failure subgroups than in the control group(F=3.65,12.52;P=0.000,0.000).According to the pairwise comparison among the chronic heart failure subgroups,the levels of serum MMP-2 and MMP-9 were significantly higher in NYHA Ⅳ grade than in NYHA Ⅲ grade,and higher in NYHA Ⅲ grade than in NYHA Ⅱ grade(all P<0.05).In patients with chronic heart failure groups,MMP-2 was positively correlated with serum BNP(r=0.866,P=0.000),and with MMP-9(r=0.516,P=0.001).Conclusions MMP-2 and MMP-9 levels might be closely correlated with chronic heart failure and show an upward trend with the progression of chronic heart failure.The levels of MMP-2 and MMP-9 are associated with BNP,which indicates that clinical monitoring of the serum level changes can provide a certain reference for diagnosis,treatment and prognosis in patients with chronic heart failure.

With the sulfate as the materials and NaOH as precipitator, Mn(0.4)Zn(0.6)Fe2O4 nanoparticles were produced, which are proved to be spinel Mn-Zn ferrite analyzed by X-ray diffraction(XRD). Their shapes are approximately global examined by transmission electron microscopy(TEM) and their average diameter is 50 nm measured with image analysis-system. The Curie temperature was measured and in vitro heating test in a alternating magnetic field was carried out. The results show that the Curie temperature is 105. 407 degrees C, While its magnetic fluid could rise to 43 degrees C - 47 degrees C due to different concentration in a alternating magnetic field. The result provide theoretical and practical evidence to select an appropriate material and concentration for tumor
Electromagnetic Fields ; Ferric Compounds ; chemistry ; Humans ; Hyperthermia, Induced ; instrumentation ; Manganese Compounds ; chemistry ; Metal Nanoparticles ; chemistry ; Microscopy, Electron, Transmission ; Neoplasms ; therapy ; X-Ray Diffraction ; Zinc Compounds ; chemistry

Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.
Escherichia coli ; genetics ; metabolism ; Flavobacterium ; enzymology ; genetics ; growth & development ; Glutathione Transferase ; biosynthesis ; genetics ; Heparin Lyase ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

Objective: To treat the depressed scars by injecting nanofat and investigate its therapeutic effect. Methods: Autologous fat was harvested from abdomen or thigh using low-pressure suction. The lipoaspirate was mechanically emulsified after rinsing. Emulsification of the fat was achieved by shifting the fat between two 5 ml syringes connected to each other by a three direct connector. After this emulsification process, the fatty liquid was again filtered over the sterile nylon cloth. Nanofat was injected into the dermis of depressed scars using a 26-gauge needle and the injection volume was 1-2 ml/cm². After three months, another injection would be performed if the depressed scar remained obvious. Results: From January 2016 to October 2017, eighteen patients and thirty-three depressed scars were treated. There was a temporary erythema of the injected area that lasted two to three weeks. The clinical result gradually improved over time and were maximal from three months postoperatively for most cases. Three months after nanofat injecting, the cavity of scars was significantly decreased; The color of scars were significantly improved and more close to the adjacent skin; The stiffness of scars was also obvious decreased. The follow-up ranged 4 months to 18 months and the average was 11.0±4.6 months. Seventeen patients were satisfied with the result, one patients was not satisfied and the satisfaction rate was 94%. No infections, fat cysts, granulomas, or other unwanted side effects were observed. Conclusions Nanofat injecting is a definite and effective treatment for depressed scars with fewer complications.

Mn0.5Zn0.5Fe2O4 nano-particles were prepared by the chemical co-precipitation, their characteristics were observed with transmission electron microscope (TEM), X-ray diffractometer (XRD) and thermal analysis system, and etc. The temperature changes of the nano-particles of Mn0.5Zn0.5Fe2O4 and its magnetic fluid explored in radiofrequency(RF,200 KHz, 4 KW) were measured. The proliferation ratio of L929 cells cultured in soak of Mn0.5Zn0.5Fe2O4 nano-particles were observed. The experiment indicates that the magnetic particles were about 40 nm diameter in average, round, had strong magnetism, and were proved to be consistent with the standard data of chart of XRD. Its magnetic fluid exposed to RF could be heated up to temperature range from 40 degrees C to 51 degrees C due to the amount of the magnetic nano-particles and intensity of the alternating magnetic field. Magnetic nano-particles were found to have no obvious cytotoxicity to L929 cells.
Animals ; Cell Line ; Ferrous Compounds ; Hyperthermia, Induced ; Magnetics ; instrumentation ; therapeutic use ; Manganese ; Materials Testing ; Mice ; Nanostructures ; Zinc

Objective To study doctor-patient interest demands satisfaction and its influencing factors of the payment system reform of the new rural cooperative medical care scheme to provide reference for the reform. Methods Cross-sectional survey was conducted from September 2016 to February 2017. Multi-stage stratified random sampling was used in six counties of three provinces in the eastern, middle and western regions of China, and mathematical statistics was applied to analyze the data. Results The doctor-patient overall interest demands satisfaction was high, but the satisfaction was lower both with the income and ability improvement of medical staff and with the benefits of farmers. The influencing factors of the satisfaction of managers in medical institutions included the type of payment, educational level and work unit (P＜0.05). The influencing factors of medical staff's satisfaction included the type of payment, work unit, and working years among others(P＜0.05). The influencing factors of farmers'satisfaction included the type of payment and the average annual income, etc(P＜0.05). Conclusions The core interest demands of both doctors and patients should be valued to enhance their satisfaction. Diseases related groups should be promoted and applied scientifically, and appropriately integrated with other methods of payment. Both doctors and patients'understanding of the payment reform should be improved by propaganda and training, to get their support and cooperation.