Objective To study the feasibility of using chitosan membrane to carry and transport melanocytes, in order to refine the technique for melanocyte transplantation with chitosan membrane. Methods Melanocytes were inoculated onto chitosan membrane and cultured for a period of time, then, electron microscopy,MTT assay and NaOH assay were carried out to estimate the adherence, growth and melanogenesis of the melanocytes. Skin wound surface was prepared in 12 nude mice, which were equally divided into 3 groups, test group inoculated with melanocytes on chitosan membrane, negative control group I treated with chitosan membrane without melanocytes, and negative control group II directly dressed immediately after the preparation of wound surface. On day 10 and 20 after the transplantation, confocal laser microscopy and immunohistochemistry were performed to observe the migration of melanocytes into the skin wound surface. Results Scanning electron microscopy and inverted microscopy showed that melanocytes were evenly distributed on and adhered well to the underlying chitosan membrane. As the growth curve of melanocytes demonstrated, chitosan membrane could support the normal growth of melanocytes, and no significant difference was observed in the synthesized melanin content between melanocytes cultured on the chitosan membrane and those in culture disks (0.087 ± 0.027 vs. 0.101 ± 0.036, t = 0.79, P > 0.05). Melanocytes were seen at the transplantation sites by confocal laser microscopy, and biopsy specimens from the transplantation sites stained positive for antimelan-A monoclonal antibody. Conclusions Melanocytes can adhere to and grow on the chitosan membrane,which can facilitate the migration of melanocytes to the transplantation sites in animals with the maintenance of biological activity of melanocytes.

BACKGROUND:Experimental studies have showed that puerarin has an obvious protective effect on osteoporosis in ovariectomized and orchiectomized mice. But the influence of puerarin in the molecular level in the process of osteoblast differentiation is seldom reported.
OBJECTIVE:To observe the effect of puerarin on the mRNA expression of alkaline phosphatase, bone sialoprotein, osteopontin and osteocalcin in osteoblasts.
METHODS:The MC3T3-E1 cells from mice cultured in vitro were randomly divided into control group, puerarin group (10-6 mol/L puerarin) and estradiol group (10-7 mol/L estradiol) to observe the effects of puerarin on the differentiation of osteoblasts. mRNA expression of alkaline phosphatase, bone sialoprotein, osteopontin and osteocalcin in MC3T3-E1 cells was determined using RT-PCR method.
RESULTS AND CONCLUSION:Puerarin and estradiol both could prolong the expression of alkaline phosphatase that reached the peak at 12 days. Puerarin and estradiol strengthened the mRNA expression of bone sialoprotein at 10 and 12 days, reduced expression of osteopontin at 5 and 12 days, and increased expression of osteocalcin at 10 and 12 days. These results reveal that puerarin can induce the differentiation of cultured osteoblasts by influencing osteoblast differentiation-related protein mRNA expressions, which may be one of the important molecular mechanisms of puerarin for prevention of osteoporosis.

In order to investigate the expression of endothelin receptor B (ETR-B) in human malignant melanoma (MM) cells A375 and SK-mel-1 and the proliferative effects of endothelin 3 (ET3) on A375 cells, RT-PCR was applied to detect the expression of ETR-B gene in human MM cells A375 and SK-mel-1. MTT method was used to evaluate the growth enhancing effects of ET3 on A375 cell line in vitro. The results showed that ETR-B gene was expressed in both MM A375 and SK-mel-1 cells. ET3 had stronger ability to enhance the proliferation of A375 cells in vitro in a concentration-dependent manner. It was suggested that ET3/ETR-B might play an important proliferative role in MM.
Cell Line, Tumor ; Cell Proliferation/*drug effects ; Endothelin-3/*pharmacology ; Melanoma/*metabolism ; Melanoma/*pathology ; Receptor, Endothelin B/*metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the therapeutic effects of adenovirus-mediated transfer of the herpes simplex virus thymidine rinase gene (HSV-tk) used by several methods and the dose-effect relationship.

METHODSDiverse doses (1 x 10(9) PFU, 1 x 10(10) PFU, 1 x 10(11) PFU) of adenovirus-mediated transfer of HSV-tk were given by intraparenchymatous, intravenous and intraperitoneal injection, and ganciclovir (GCV) (100 mg.kg(-1).d(-1)) was injected into the cavity of the peritoneum to treat human hepatocarcinoma. The change of tumors size was observed and the fragments of HSV-tk gene were tested.

RESULTSIn nude mice after intraparenchymatous injection and high-dose (1 x 10(11) PFU) intravenous injection, the tumors were suppressed significantly (t = 13.1, 12.4, P < 0.01) and lots of fragments of HSV-tk gene were observed. In mice after intraperitoneal injection and low-dose (1 x 10(9) PFU, 1 x 10(10) PFU) intravenous injection, no suppressive effect was observed (t = 1.8, 1.0, 2.1, 1.1, 0.8, P > 0.05) with few or without fragments in the tumors.

CONCLUSIONSAdenovirus-mediated transfer of the HSV-tk by intraparenchymatous or intravenous injection is effective in treatment of hepatocarcinoma in nude mice, but intraperitoneal injection has no therapeutic effect.

Adenoviridae ; genetics ; Animals ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Humans ; Liver Neoplasms, Experimental ; therapy ; Mice ; Mice, Inbred BALB C ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; Tumor Cells, Cultured

OBJECTIVETo investigate in vitro and in vivo bystander effect, including distance bystander effect of adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene (HSV-tk).

METHODSIn vitro, mixed tk + BEL-7402 cells and tk-BEL-7402 cells in diverse proportions and ganciclovir (GCV) was given, then tested the survival ratio of cells by MTT. In vivo, 5 x 10(6) and 5 x 10(7) tk + BEL-7402 cells were injected into the tumors in nude mice following GCV. The change of the size of the tumors was observed. To observe distance bystander effect, adenovirues with HSV-tk (1 x 10(9) PFU) were injected into the tumor, which was the one of the bilateral tumors in nude mice. Subsequently, GCV was given and the change of the tumor was observed.

RESULTSSignificant bystander effect was observed in vitro and in vivo. In vitro when tk + cells: tk(-) cells was 1:9, the survival rate of mixed cells was 36.6%. When the proportion of tk(+) cells was 90%, the survival rate of mixed cells was 3.2%. In vivo, those tumors with injection of tk(+) BEL-7402 cells were suppressed (P < 0.05). But in group of distance bystander effect the tumors on another side were not suppressed.

CONCLUSIONIn vitro, bystander effect exists. In nude mice, if tk(+) cells and tk(-) cells are contiguous, bystander effect is significant, or probably no bystander effect.

Adenoviridae ; genetics ; Animals ; Bystander Effect ; Disease Models, Animal ; Ganciclovir ; therapeutic use ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Neoplasms, Experimental ; drug therapy ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism ; Tumor Cells, Cultured

To get specific scFv (Single-chain fragment variable) antibody against soluble Abeta1-42(Amyloid-beta) oligomers, we constructed a human single-chain Fv (scFv) antibody library by phage display technology. Using RT-PCR, we amplified the variable heavy (VH) and variable light (VL) genes from peripheral blood lymphocytes (PBL). Then we obtained the scFv fragments through SOE-PCR, and the scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent Escherichia coli TG1 cells. Consequently, a scFv phage display library containing 2.5 x 10(9) clones was constructed. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for Abeta1-42 oligomers were enriched after four rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. Positive clone B19 was used to infect E. coli HB2151 to express soluble scFv antibody. SDS-PAGE and Western blotting analysis showed that the soluble scFv B19 antibody was expressed successfully and could bind specifically to Abeta1-42 trimer and protofiber. The specific scFv against Abeta1-42 oligomers can be used in the therapeutic research on Alzheimer's disease.
Amyloid beta-Peptides ; genetics ; immunology ; Antibody Specificity ; immunology ; Humans ; Peptide Fragments ; genetics ; immunology ; Peptide Library ; Single-Chain Antibodies ; genetics ; immunology ; isolation & purification

OBJECTIVETo identify protein-protein interaction partners of PER1 (period circadian protein homolog 1), key component of the molecular oscillation system of the circadian rhythm in tumors using bacterial two-hybrid system technique.

METHODSHuman cervical carcinoma cell Hela library was adopted. Recombinant bait plasmid pBT-PER1 and pTRG cDNA plasmid library were cotransformed into the two-hybrid system reporter strain cultured in a special selective medium. Target clones were screened. After isolating the positive clones, the target clones were sequenced and analyzed.

RESULTSFourteen protein coding genes were identified, 4 of which were found to contain whole coding regions of genes, which included optic atrophy 3 protein (OPA3) associated with mitochondrial dynamics and homo sapiens cutA divalent cation tolerance homolog of E. coli (CUTA) associated with copper metabolism. There were also cellular events related proteins and proteins which are involved in biochemical reaction and signal transduction-related proteins.

CONCLUSIONIdentification of potential interacting proteins with PER1 in tumors may provide us new insights into the functions of the circadian clock protein PER1 during tumorigenesis.

Base Sequence ; Cell Line, Tumor ; Escherichia coli ; genetics ; metabolism ; Humans ; Molecular Sequence Data ; Neoplasms ; genetics ; metabolism ; Period Circadian Proteins ; genetics ; metabolism ; Protein Binding ; Proteins ; genetics ; metabolism ; Two-Hybrid System Techniques

Objective: To account the direct cost of uterine cervix carcinoma treatment in China and to explore the related factors which influence the direct financial burden of the disease. Methods: Data was collected through the medical record system and telephone interviews in 14 county-level hospitals and 9 provincial and municipal hospitals from 14 provinces/municipalities enrolled in the Chinese National Health Industry Research Project in 2015. The direct financial burden of uterine cervix carcinoma treatment consisted of the direct medical cost and the direct non-medical cost of treatment in different pathological cervical cancer stages and precancerous lesions. Multiple liner regression method was used to analyze the factors affecting the costs. Results: The age of the 3 246 patients was (46.40±10.43) years, including 2 423 patients from provincial and municipal hospitals and 823 patients from county-level hospitals. The direct financial burden for one patient of pathological uterine cervix carcinoma stage or precancerous lesion ranged from 10 156.3 yuan to 75 716.4 yuan in provincial and municipal hospitals, and for patients from county-level hospitals, the cost was between 4 927.9 yuan and 47 524.8 yuan per person. There was a wide gap between the direct financial burden of patients in different disease stages. The direct financial burden of patients with precancerous lesions ranged from 4 927.9 yuan per person to 11 243.0 yuan per person, as for patients of pathological uterine cervix carcinoma stages, the direct financial burden was between 29 274.6 yuan and 75 716.4 yuan per person. The factors which influence direct financial burden would include: the levels of the hospital, pathological period, medicare reimbursement, days of treatment, and the methods of treatment (P<0.001). Conclusion The direct financial burden of diseases in patients with pathological uterine cervix carcinoma stage or precancerous lesion differed in different levels of hospital and pathological periods. In addition, medicare reimbursement, days of treatment, and the methods of treatment all had impact on it.

Objective:To explore the effect of foraging exercise (FE) on the behavior of rats with post-stroke depression (PSD) and the expression of 5-Hydroxytryptamine 1A (5-HT1A) receptor and transforming growth factor β1 (TGF-β1) in their frontal lobes.Methods:Thirty-six healthy male Sprague-Dawley rats were randomly divided into an ischemia-reperfusion (I/R) group, a PSD group and a PSD+ FE (FE) group, each of 12. The right middle cerebral artery of each was occluded using the thread occlusion method with 1.5h of ischemia. In the PSD and FE groups, mild stimulation was administered at unpredictable intervals over 3 weeks beginning 1 week after the successful modeling. The rats in the I/R group were raised in a group. Those in the PSD group were raised in individual cages. Those in the FE group were raised in a single cage and foraged freely for a total of 4 weeks. Four and eight weeks after the modeling, the body weights were measured, and the open field, social interaction (SIT) and sugar preference tests were administered to all of the groups. Four weeks later, all of the rats were sacrificed and their brains were sliced and stained. The expression of 5-HT1A receptor and TGF-β1 in the frontal lobe was detected using western blotting.Results:One week after modeling, there was no significant difference in average body weight or the average behavioral scores among the three groups. After four weeks the PSD and FE groups had significantly lower average body weight than the I/R group, fewer counts of rearing and grid crossing, longer SIT latency, less interaction time and lower average sugar preference (all significant differences). After eight weeks the average body weight had increased in each group. SIT latency had shortened and interaction time had increased in the FE group, and the rearing and grid crossing counts and sugar preference had increased in the PSD and FE groups. At that point the FE group had significantly greater average body weight than the PSD group, more counts of rearing and grid crossing, shorter SIT latency, increased interaction time, and greater sugar preference. The ratio of residual brain volume in the right hemisphere of the PSD and FE groups was significantly lower on average than in the I/R group. However, there was no significant difference in the right residual brain volume ratio between the PSD and FE groups. Staining revealed that the pathological changes in the frontal lobes of the FE group had been significantly relieved compared with the PSD group. Eight weeks after the operation the increases in average 5-HT 1A receptor and TGF-β1 levels in the FE group were significantly greater than in the PSD group.Conclusion:Foraging can relieve the depressive symptoms of rats modeling post-stoke depression. The mechanism may be related to alleviating the pathological damage and increasing the expression of 5-HT1AR and TGF-β1 in the frontal lobe. Early chronic stress may increase the volume of cerebral infarction, at least in rats.

Objective:To explore the effects of foraging exercise (FE) on depressive-like behaviors and expression of transforming growth factor-β1 (TGF-β1) in hippocampus of rats with ischemic stroke after chronic stress.Methods:The right middle cerebral artery occlusion (MCAO) model was used in 30 male adult clean grade SD rats by suture method.According to the body weight, rats were evenly divided into stroke group ( n=10) and chronic unpredictable mild stimulation (CUMS) group ( n=20). Rats of CUMS group received stress induction 1 week after operation and lasted for 3 weeks. Then, according to random number generator of SPSS 24.0 software, the depression rats were divided into post-stroke depression (PSD) group( n=10) and FE groups ( n=10). The FE group received free FE intervention for 4 weeks. Body weight, water maze test, novelty inhibition feeding test (NSFT) and sucrose preference test (SPT) were performed at the end of the 1st, 4th and 8th week, respectively. The expression of TGF-β1 in hippocampus was detected by Immunohistochemistry (IHC) and Western blot (WB), and the levels of TGF-β1 and TNF-α in serum were detected by ELISA. SPSS 24.0 software was used for statistical analysis. The behavioral data were compared by two factor repeated measurement analysis of variance. One way ANOVA was used for comparison among groups, and LSD test was used for further pairwise comparison. Results:(1) The interaction between group and time had statistical significance on body weight, latency and food intake of NSFT and sucrose preference index(SPI) ( F=2.936-12.098, all P<0.05). After 4 weeks, compared with the stroke group((343.80±19.34)g, (12.10±6.97)s, (0.75±0.09)%), the body weight((307.80±17.23)g, (305.30±24.39)g), and SPI((0.52±0.06)%, (0.53±0.07)%) of PSD group and FE group were lower and the NSFT latency((21.70±7.02)s, (22.40±0.84)s) was longer (all P<0.05). After 8 weeks, SPI in FE group was higher than that in PSD group ( P=0.045). There were significant differences in body weight of three groups, NSFT latency and SPI of PSD group and FE group, and food intake of stroke and FE group ( F=8.478-196.548, all P<0.05). There was no interaction between group and time in the water maze test. Main effect of time ( P=0.034) and main effect of group ( P<0.01) had statistical significance on escape latency. The escape latency after 4 weeks was longer than that after 1 week ( P=0.003). The latency of PSD group was longer than that of stroke group ( P=0.005), and latency of FE group was shorter than that of the PSD group ( P<0.01). The main effect of group had statistical significance in the number of crossing quadrant ( P<0.01). The number of crossing quadrant of FE group was less than that of PSD group ( P<0.01). (2) Immunohistoche mistry staining showed that compared with the stroke group, the expression of TGF-β1 was down-regulated in 3 areas of hippocampus of PSD group (CA1, CA3 and DG) ( t=5.449-9.353, all P<0.01). Compared with stroke group, the expression of TGF-β1 of CA1 ( t=7.433, P<0.01) in FE group was down-regulated, but was up-regulated in CA3 ( t=3.342, P<0.05) of FE group. Compared with the PSD group, the expression of TGF-β1 was up-regulated in CA3 and DG of FE group ( t=7.811, 8.790, both P<0.01). (3) Western blot results: Compared with stroke group, the expression of TGF-β1 in hippocampus of PSD group was down-regulated ( t=3.255, P<0.01). Compared with the PSD group, the expression of TGF-β1 in hippocampus of FE group was up-regulated ( t=2.906, P<0.05). (4) ELISA detection showed that compared with the stroke group, the levels of TGF-β1 decreased ( t=2.224, P<0.05), but TNF-α increased ( t=6.127, P<0.01) in PSD group.Compared with the PSD group, the expression of TGF-β1 in FE group increased significantly ( t=4.417, P<0.01). Conclusion:Foraging exercise can improve the depressive behavior symptoms of ischemic stroke rats after chronic stress, and its mechanism may be related to the increasing expression of TGF-β1, which can alleviate the inflammatory reaction in hippocampus.