Objective To obtain skin seed cells which can highly express active vascular endothelial growth factor 165(VEGF165) so as to promote refractory wound healing by gene therapy in combination with cell engineering.Methods After pIRES2-EGFP-hVEGF165 was transfected into dermal mesenchymal stem cells(DMSCs) by lipofectin,the expression of VEGF mRNA was detected by RT-PCR,VEGF protein in the supernatant and in the cells were assayed by enzyme-linked immunosorbent assay(ELISA) and Westen blotting respectively.To evaluate the activity of VEGF secreted by transfected DMSCs,hECV304 was cultured with the supernatant of transfected DMSCs and its proliferation activity was analyzed by MTT assays.Meanwhile,the proliferation activity of VEGF-transfected DMSCs and non transfected DMSCs was investigated by MTT.Results The results of RT-PCR,ELISA and Western blot demonstrated that the expression of VEGF in transfected DMSCs was about 1.6 times than that of control DMSCs.The product not only enhanced the proliferation of hECV304 but also increased the proliferation of transfected DMSCs.Conclusion The plasmid pIRES2-EGFP-hVEGF165 is successfully transfected into DMSCs with the aid of lipotransfection,and hVEGF165-transfected DMSCs might high-efficiently secrete highly active VEGF165.

Objective Non-muscle myosin heavy chain 9 (MYH9) plays an important regulatory role in the development of tumor.This study aimed to explore the expression of MYH9 in osteosarcoma tissues and its effects on epithelial-mesenchymal transition and invasion of osteosarcoma cells.Methods We collected 52 cases of osteosarcoma tissues and para-carcinoma tissues at 5 cm form the edge of the tumor.RT-PCR and immunohistochemistry were used to analyze the expression level of MYH9 mRNA and protein in the osteosarcoma tissues and para-carcinoma tissues.MYH9 shRNA plasmid was transfected into U2-OS cells to silenced the expression of MYH9, after transfected, the cells were divided into three groups: the normal U2-OS cells were the control group, the U2-OS cells transfected with empty plasmid were the empty group and U2-OS cells transfected with MYH9 shRNA were interference group.RT-PCR was used to detect the changes of MYH9 mRNA levels in the U2-OS cells, the protein level of MYH9, EMT related protein E-cadherin and Vimentin were detected by Western blot, and the ability of cell invasion was evaluated by Transwell assay.Results The results of RT-PCR showed that the relative expression MYH9 mRNA in para-carcinoma tissues(1.526±0.148) was significantly lower than that in cancer tissues (3.547±0.195) (P<0.05).The results of immunohistochemistry showed that MYH9 protein was mainly expressed in cytoplasm, and the expression in cancer tissues was significantly higher than that in para-carcinoma tissues, the positive expression rate were 59.6%(31/52) and 26.9%(14/52) respectively, the difference was statistically significant(P<0.05).The results of Western blot showed that the relative expression of MYH9 mRNA in interference group was significantly lower than that in control group and empty group (P<0.05) after silenced MYH9 gene, and compared with the control group, the E-cadherin in U2-OS cells was significantly up-regulated but the Vimentin was down-regulated.After 48h, all of the groups had cells through the microfiltration membrane, the numbers of cells through the microfiltration membrane in interference group(41.2±15.1) was significantly lower than that in control group(117.3±12.4) and empty group(193.5±14.7) (P<0.05).Conclusion The expression of MYH9 protein in osteosarcoma tissues was significantly higher than that in para-carcinoma t tissues, silenced MYH9 gene can reduce the invasive ability of osteosarcoma by reducing the epithelial interstitial transition.

Objective To detect whether mice bone marrow mesenchymal stem cells(BMSCs)can contribute to the regeneration of hepatocytes in bone marrow chimeric mice.Methods Female recipient mice(C57BL/6J)underwent whole body gamma-ray irradiation with a dose of 10 Gy to ablate their bone marrow,followed by immediate tail vein injection of BMSCs isolated from male GFP transgenic mice.Animals were killed at different phase points:1 week,1 month,and 3 months.Using fluorescence microscope we directly observed GFP-positive cells in the liver frozen sections,and we also prepared the parafilm sections to detect the GFP-positive cells and the coexpression of GFP and Alb,CK18 by immunohistochemistry and immunofluorescence respectively.Results We found numerous GFP-positive cells in recipient mice liver at 1 week after BMSCs transplantation,some at 1 month and seldom at 3 months.There were some cells coexpressing GFP and Alb,CK18 at all the phase points.Conclusion Allotype BMSCs can differentiate into Alb and CK18 positive hepatocytes in bone marrow chimeric mice,which will become an ideal cell resource for liver tissue project.

Objective To examine the expression of human ?-defensin 2 (hBD_ 2 ) recombinant adenovirus expression vector in rat dermal multipotent stem cells (dMSCs) and to observe the antiseptic activity of recombinant hBD_ 2 . Methods The expression of hBD_ 2 in dMSCs was examined by RT-PR, fluorescent immunochemistry and Western blotting, and the concentration of recombinant hBD_ 2 in supernate was measured by ELISA. The antiseptic activity of recombinant hBD_ 2 was assessed by K-B disc agar diffusion test. Results hBD_ 2 could be effectively expressed in dMSCs, and the concentration of recombinant hBD_ 2 in supernate was about 743.6 ng/ml . Recombinant hBD_ 2 in supernate showed antiseptic activity. Conclusion Recombinant adenovirus expression vector of hBD_ 2 could be effectively expressed in dMSCs, and the recombinant hBD_ 2 in supernate showed obvious antiseptic effects toward some standard bacteria lines.

Objective To observe the repair effect of human ?-defensin 2 (hBD 2)-modified rat dermal multipotent stem cells (dMSCs) transplantation on infected wound. Methods Thirty Wistar rats were excised a piece of whole-layer back skin, 3 mm in diameter, then infected the wound with 1?10 8/ml pseudomonas aeruginosa 1 ml, then the rats were injected on the wounded back respectively with dMSCs modified by hBD 2 (n=10), or pure dMSCs (n=10) or none as control (n=10). The repair effect was evaluated by observing the amount of bacteria under the scar, wound healing time and the percentage of remaining wound area. Results The amount of bacteria under the scar in rats that were transplanted with dMSCs modified by hBD 2 was less than that in rats transplanted with dMSCs or controls (P

Zebrafish is a novel model organism for drug metabolism research because of its small size, easy feeding, low reproduction cost and high reproduction rate.This paper outlines the construction of zebrafish metabolic model, illustrating the advantages of zebrafish model with high genetic similarity to human, well-developed enzyme system, and low substrate effect; and then reviews the application of zebrafish model in the synthesis of various new psychoactive substances such as cannabinoids, fentanyl, piperazines, and synthetic cathinones.The common metabolic reactions in zebrafish, including phase I (oxidation, N-demethylation, O-deethylation, hydroxylation and N-desalkylation) and phase II (sulfation and glucuronidation) reactions, are summarized, and the differences between the metabolites of zebrafish and those of real human body fluid samples are mainly related to the types of enzymes.This paper also points out the great potential and further research trends of zebrafish model in the study of new psychoactive substances.

OBJECTIVETo study the effects of dosages of total body irradiation on the healing process of cutaneous wounds and to observe the changes of wound area at different periods after injury.

METHODSThe entire body irradiation from a (60)Co gamma-ray source was performed on Wistar rats. The single dosage varied from 1 to 8 Gy. Within 1 h after irradiation, two whole thickness circular cutaneous wounds corresponding to 2.5% of total body surface area (Phi = 22 mm) were produced on the back of the animals (combined injury groups). Same wounds were produced on rats with no irradiation (single wound group). Wound healing was observed at different points after injury.

RESULTSAfter total body irradiation with the dose of 1, 2, 3, 4, 5, 6, 7 or 8 Gy, the wound healing was obviously retarded as the dosages increased. The wound area remained was larger in the large dosage groups than in the small dosage groups. Seven days after injury, there was 33.5% wound surface left unhealed in the single wound group, whereas in the combined injury groups, 35.4%, 38.1%, 41.6%, 48.8%, 53.9%, 63.7%, 69.2% and 73.9% of the wound surfaces remained unhealed, respectively. Statistical analysis showed marked correlations between the various times after total body irradiation and various dosages to the percentage of unhealed wound surface. Nine dose-effect relation formulae were deduced according to the statistical results.

CONCLUSIONSIn soft tissue trauma combined with radiation injury, the delay of wound healing is related to the dose of radiation inflicted. It is also related to the time between injury and time of observation.

Animals ; Dose-Response Relationship, Radiation ; Female ; Male ; Rats ; Rats, Wistar ; Time Factors ; Whole-Body Irradiation ; Wound Healing ; radiation effects

Background Hypertension is influenced by both genes and environment. At present, most studies on the relationship among occupational stress, polymorphisms of ATP binding cassette subfamily A member 1 (ABCA1) or angiotensinogen (AGT) genes, and hypertension focus on single gene or single environmental effects. Objective To investigate the relationship of potential interactions between ABCA1 and AGT gene polymorphisms and occupational stress with the prevalence of hypertension. Methods A total of 198 hypertensive patients were selected as the case group from the 1200 oilfield workers in Karamay Oilfield in 2018 with random cluster sampling method, and the control group was selected as 1∶1 matched subjects for sex, age (±3 years), and ethnicity, after excluding blood samples, questionnaires, or DNA purity (concentration) that did not meet the inclusion criteria. Finally, 153 workers in the hypertension case group and 153 workers in the control group were determined. A questionnaire was used to collect general information of the oilfield workers, and the Occupational Stress Inventory Revised Edition (OSI-R) was used to evaluate occupational stress. Polymerase chain reaction-restriction fragment length polymorphism technology was used to detect the genotypes of V825I and R219K loci of ABCA1 as well as M235T and T174M loci of AGT. The gene-gene interaction of ABCA1 and AGT and the relationship between the interaction of gene-occupational stress and the prevalence of hypertension were analyzed by generalized multi-factor dimensionality reduction method. Results The difference of reported occupational stress between the hypertension case group and the control group was statistically significant (P＝0.001), and the reporting rate of high occupational stress in the case group (65.4%) was higher than that in the control group (47.7%). The genotype and allele distributions of ABCA1 V825I, ABCA1 R219K, and AGT M235T between the hypertension case group and the control group were significantly different (P<0.05). The results of conditional logistic regression analysis showed that VI and II genotypes at V825I locus of ABCA1 (OR_VI=1.682, 95%CI: 1.099-2.573; OR_II=1.708, 95%CI: 1.045-2.790), TT genotype at M235T locus of AGT (OR=1.645, 95%CI: 1.022-2.647), and high occupational stress (OR=2.642, 95%CI: 1.228-5.686) increased the risks for hypertension (P<0.05). There was no significant correlation between ABCA1 R219K or AGT T174M polymorphisms and the prevalence of hypertension (P>0.05). The gene-gene interactions between ABCA1 V825I and R219K loci and AGT M235T locus were associated with hypertension (accuracy on training and test sets was 0.68 and 0.63, respectively, with a cross-validation coefficient of 10/10, P<0.05), and ABCA1 V825I locus positively interacted with AGT M235T locus. The gene-environment interactions among ABCA1 V825I and R219K loci, AGT M235T locus, and occupational stress were associated with hypertension (accuracy on training and test sets was 0.74 and 0.63, respectively, with a cross-validation coefficient of 10/10, P<0.05), and AGT M235T locus negatively interacted with occupational stress. Conclusion Genotype VI and II of V825I locus at ABCA1, genotype TT of M235T locus at AGT, and high occupational stress may be risk factors for oilfield workers’ hypertension in Karamay, and the interactions of gene-gene and gene-environment among ABCA1 and AGT gene polymorphisms and occupational stress may be associated with hypertension.

OBJECTIVE：To prepare paclit axel and schisandrin B liposomes modified by cell penetrating peptide RPV ，and to preliminarily evaluate its anti-tumor activity in vitro . METHODS ：RPV modified paclitaxel and schisandrin B liposomes were prepared by film dispersion method. Box-Benhken design-response surface methodology was used to optimize the prescription technology of RPV modified paclitaxel and schisandrin B liposomes using the amount of cholesterol and paclitaxel ，the time interval of ultrasound probe as factors ，average entrapment efficiency of paclitaxel and schisandrin B was used as the index. The liposomes prepared by the optimal technology were characterized. Sulfonylrhodamine B staining method was used to investigate in vitro toxicity of RPV modified blank liposomes ，paclitaxel and schisandrin B liposomes ，RPV modified paclitaxel and schisandrin B liposomes to human ovarian cancer cell SK-OV- 3. The effects of 3 kinds of liposomes on the migration and invasion ability of SK-OV-3 cells were investigated by cell scratch test and Transwell chamber invasion test. RESULTS ：The optimal prescription technology was phospholipid 44 mg，cholesterol 8 mg，paclitaxel 0.64 mg，schisandrin B 1.5 mg，ultrasonic probe time interval 5 s，prescription dosage 5 mL. According to the optimal prescription technology ，the liposomes were spherical in shape ，and the particle size was （126.49±1.19）nm，Zeta-potential was （－4.83±0.61）mV，average entrapment efficiency of liposomes was （93.88±1.67）％. Compared with RPV modified blank liposomes ，after treated with paclitaxel and schisandrin B liposomes and RPV modified paclitaxel and schisandrin B liposomes ，the survival rate ，migration inhibition rate and invasion rate of SK-OV- 3 cells were significantly decreased （P＜0.05）. The effects of RPV modified paclitaxel and schisandrin B liposomes was better than those of paclitaxel and schisandrin B liposomes （P＜0.05）. CONCLUSIONS ：RPV modified paclitaxel and schisandra B liposome are successfu lly prepared ，and they have certain antitumor activity in vitro .