Objective Compare the reproductive activity of pancreatic stem cells isolated from diabetic rat with that of normal rat to assess the effect of microenvirement on stem cells'proliferation.Methods Diabetic rat model were prepared by peritoneal injecting 60mg/kg streptozotocin.Pancreata from 10 diabetic rats and 10 normal rats were trimmed and digested into single cells which were then inoculated onto the culture plate respectively.After passaging 3 generation the cells were identified by nestin immunostaining.During culturing the cell growth curve was observed and the cell cycle of the third and seventh generation cultured cells was evaluated by flow cytometry.Results After passaging pancreatic stem cells was purified and identified by nestin immunostaining.The reproductive activity of diabetic rat was much better[ratio of s stage:(20.7?2.7)% vs(14.1?2.5)%,P0.05].Conclusions The stem cells are stimulated to proliferate after islet injure.The reproductive activity of cells can be provoked by the microenvironment.

Am investigation was done to identify the best and safe dosage of magnesium isoglycurrhizinate in treating hepatitis B.All 1 50 cases suffering from hepatitis B were randomly divided into five groups as A,B,C,D,E.Cases in group A were treated with magnesium isoglycurrhizinate in 100 mg per day for two weeks sololy,and in group B,C,D,E in 150 mg,200 mg,250 mg and 300 mg respectively.The changes of symptoms,index of hepatic function,clinical effective rates and side-effects were observed from treatment beginning to the end.The results that revealed there were no different effects among groups B to E,but less therapeutic effects in group A,and no obvious side-effects in all groups,suggesting that 150 mg dosage of magnesium isoglycurrhizinate should be a safe and the best dosage for treating hepatitis B.

Objective To study the immunoreaction of the recombinant proteins encoded by the fragments of ORF2 ( second open reading frames) gene of hepatitis E virus ( HEV). Methods The aimed sequence from the full-length ORF2 in clone PEH2 , which was derived from a Chinese strain of HEV,was amplified and cloned it into vector pcDNAS. 1 which was then transfected to 293 cell. Results The ORF2 protein was present in the soluble fractions of the cell lysate. The expressed protein of HEV ORF2 in 293 cells by using a plasmid pcDNA3. 1-based system showed positive on immunoblots probed against antibodies raised in BALB/c mice. Conclusion The experimental results laid a foundation for developing diagnostic reagent to detect HEV by using the expressed products of ORF2 protein.

Objective:To investigate the effect of licochalcone A on osteoarthritis in rats and its relationship with p38-MAPK inflammatory signaling pathway.Methods:A total of 160 male Wistar rats were randomly divided into blank non-intervention, blank intervention, arthritis non- intervention and arthritis intervention groups with 40 rats in each group. Rats in the arthritis groups were subjected to unilateral anterior cruciate ligament transection, while rats in the blank groups were only subjected to skin incision and suture. Rats in the intervention groups were treated by intra-articular injection of 1 mL 10 μmol/L licochalcone A for 8 successive weeks. Eight weeks later, the cartilage of rats in each group was stained with safranin, and osteoarthritis soft tissue was scored according to Osteoarthritis Research Society International guideline under the optical microscope. The cartilage was cultured in low glucose cell culture medium supplemented with 5% fetal bovine serum for 48 hours. The contents of nitric oxide, prostaglandin E 2, sulfated glycosaminoglycan and collagen II in the medium were determined by the chemiluminescence reaction method. The expression levels of p38, phosphorylated p38 (p-p38) and matrix metalloproteinase in cartilage tissue were detected by western blot assay. Results:The progress of osteoarthritis in rats treated with licochalcone A was slow. The Osteoarthritis Research Society International score in the arthritis intervention group was significantly lower than that in the arthritis non-intervention group [(3.8 ± 1.7) points vs. (9.7 ± 1.2) points, P = 0.0064]. The contents of nitric oxide, prostaglandin E 2, sulfated glycosaminoglycan, and collagen II in the arthritis intervention group were (77.84 ± 17.65) μmol/mg and (6.78 ± 1.76) ng/mg, (89.78 ± 9.76) μg/mg, and (1.78 ± 0.76) μg/mg, respectively, which were significantly lower than those in the arthritis non-intervention group [(107.56 ± 18.74) μmol/mg, (10.756 ± 1.87) ng/mg, (125.75 ± 8.87) μg/mg, (3.76 ± 0.88) μg/mg, (NO: P = 0.002; PGE 2: P < 0.001; sGAG: P < 0.001; Collagen II: P < 0.001). Western blot assay results revealed that the relative expression of p38, p-p38, p-p38 to total p38 ratio, matrix metalloproteinase in the arthritis intervention group were (3 454 ± 421), (2 072 ± 175), (0.65 ± 0.14 )and (1 776 ± 765), respectively, which were significantly lower than those in the arthritis non-intervention group (5 322 ± 323), (4 257 ± 184), (0.89 ± 0.11), (3 865 ± 874)( p38: P < 0.001; p-p38: P < 0.001; p-p38/p38: P = 0.002; MMP: P = 0.001). Conclusion:Licochalcone A can delay the progression of osteoarthritis in rats with osteoarthritis through inhibiting inflammatory reaction and cartilage matrix degradation, and p38-MAPK signaling pathway may be involved in the regulation process.

OBJECTlVE To study the anti-HBV activity of prepared recombinant human serum aIbu-min-interferon α-2b fusion protein(HSA-IFNα-2b) in vitro. METHODS HepG2 ceIIs were infected with recombinant adenovirus with green fIuorescence protein and 1.6-foId HBV DNA(AdGFP-HBV). The ex-pression of HBV antigens,HBsAg and HBeAg in cuIture medium was detected by ELISA assay. The tox-icity of HSA-IFNα-2b on HepG2 ceIIs was evaIuated by mTT assay.The reIative expression of HBV RNA in ceIIs and the absoIute quantity of HBV DNA in cuIture supernatant were determined by quantitative PCR assay. The activity of HBV enhancer Ⅰ was detected by DuaI-Reporter gene assay. RESULTS HBV couId repIicate and express in HepG2 ceIIs after infection with AdGFP-HBV. The expression of HBsAg and HBeAg in cuIture serum of HepG2 ceIIs infected with AdGFP-HBV decreased by 51.32%(P﹤0.01)and 50.26%(P﹤0.01),respectiveIy,when HSA-IFNα-2b 500 kU·L-1 was added. The same concentration of HSA-IFNα-2b didn't inhibit the proIiferation of HepG2 ceIIs,but inhibited HBsAg in a concentration-dependent manner. The regression formuIa between HBsAg inhibitory rate(Y)and con-centration of HSA-IFNα-2b(X)was Y=21.11 IgX+11.91(r 2 = 0.954),IC50 = 63.76 kU·L-1 . HBV RNA in ceIIs and HBV DNA in the cuIture serum decreased by 52.83%(P﹤0.01)and 53.07%(P﹤0.01), respectiveIy,when HSA-IFNα-2b 500 kU·L-1 was added. The activity of enhancer Ⅰ decreased by 40.04%(P﹤0.01)when HSA-IFNα-2b 500 kU·L-1 was added. CONCLUSlON The ceII modeI of HBV repIication for evaIuating anti-HBV agents is successfuIIy estabIished. HSA-IFNα-2b exhibits noticeabIe anti-HBV effect invitro.

Objective To investigate the optimal concentration of manganese-enhanced MRI (MEMRI)in the visual pathway of experimental rats.Methods Sprague-Dawley rats were intravitreally injected with 3 μL of 10 - 100 mmol/L MnCl2 ,respectively. The contrast-to-noise ratio (CNR)of MEMRI for optic nerve(ON)and midbrain superior colliculus (SC)enhancement were measured at differ-ent concentrations of MnCl2 .Results The ON and SC were all clearly detected by MEMRI 24 h after unilateral intravitreal injected 10-100 mmol/L MnCl2 ,respectively.The CNR increased with the increasing concentration of MnCl2 from 10 to 50 mmol/L;But the CNR decreased from 50 to 100 mmol/L.The enhancement of superior colliculus were higher than optic nerve.Conclusion The optimal concentration of MnCl2 is 30 mmol/L(3 μL)through intravitreal injection for the rat visual pathway on 1.5T MEMRI.

Objective To study the effect of the uncellular tissue engineering complexes of autolegous red bone marrow wrapped by facial flap with vessels in repair of large segment bone defect infected with low virulence bacteria so as to provide evidence for the clinical application. Methods The study included 38 cases of limb bone defect infected with low virulence bacteria after trauma.Autologous red bone marrow (ARBM) was taken to prepare uncelluar tissue-engineered complexes with osteoinductive absorbing material (OAM) containing bone morphogenetic protein (BMP).A facial flap with capillary network originating from an anonymous vessel adjacent to the bone defect was prepared to wrap the tissue engineered bone and fill the bone defect.Pathological focus clearance and tissue-engineered complexes compounded with ARBM implantation were performed in 18 cases (Group A) and pathological focus clearance and tissue-engineered complexes of autolegous red bone marrow wrapped by facial flap with vessels implantation in the other 20 cases ( Group B).The blood routine and supersensitive CRP were examined to monitor the inflammation reaction; X-ray was used to observe the bone defect repair; histology and bacteriology examinations were performed in partial cases at 3,6,12,18 months after operation. Results Six months after operation,5 cases of Group A were infected and the bacteria cultivation was as positive as that before the operation.The histological observation at ( 14.0 ± 0.5 ) months after operation showed that fibrous connective tissues between the bone fracture ends existed in the pathological area in 10 cases,of whom four cases were filled with inflammatory fibrous granulation tissues and few dead bones in the pathological area,and the bacterial examination was positive.There was no infection in Group B after operation.The histological observation manifested periosteum like tissues formation from the primary facial flap,mature bone structure formation in the primary pathological area and non-inflammatory infiltration in 16 cases and the bacteria cultivation was negative in these cases.The external fixation frame was taken out (12.2 ± 0.3 )months after operation because the synostosis appeared and the structure was stable in the other seven cases including three cases in Group A and four in Group B and the histological and bacterial examination were not performed.At each time point after operation,not only the blood routine but also the supersensitive CRP and the X-ray quantification grade of Group B were significantly more than those of Group A (P ＜ 0.05 ). Conclusions The uncellular tissue-engineered complexes of autolegous red bone marrow wrapped by facial flap with vessels is a feasible method for repairing the infected bone defect by first intention,since it can resist infection,obviously promote the bone recovery and advance the quality and quantity of osteanagenesis.

Objective To obtain Chinese hamster ovary (CHO) cell lines that stably express a targeting complement inhibitor CR2-CD59.Methods The recombinant plasmid PEE14.1-CR2-CD59 was constru-cted by cloning the DNA fragment CR2-CD59 into plasmid PEE14.1,and the obtained plasmid was transfected into CHO cells by FuGENE 6.The clones with stable high expression of target fragment were selected by methionine sulfoximine (MSX),the expression of CR2-CD59 was analyzed by ELISA,SDS-PAGE and Western blotting analysis.Results Several stable expression clones were obtained,and CR2-CD59 was highly expressed in the secret form in CHO cells.SDS-PAGE analysis showed that the molecular weight of the recombined protein CR2-CD59 was consistent with the predicted one.ELISA and Western blotting results revealed that the CR2-CD59 could react with both anti-human CR2 and anti-human CD59 polyclonal antibodies.Compared with serum-containing medium,the protein was highly expressed in serum-free medium (P

A total of 38 cases of senile brucella spondylitis disease at our hospital during January 2002 and March 2012 were analyzed .After admission , all of them were definitely diagnosed on the basis of epidemiological history , clinical manifestations , laboratory tests , imaging and pathological examinations . Over a follow-up period of 12 months, 17 cases were cured after standardized drug treatment .Among 21 surgical cases, there were curing (n=17) and improving (n=2).Senile brucellosis spondylitis has distinct serological and pathological characteristics .And formulating the diagnostic criteria may improve its diagnostic rate and reduce its misdiagnostic rate .And standardized drug therapy achieves a better curing rate and a proper timing of surgical intervention improves its clinical outcomes .

To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5' phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E. coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes.
Amino Acid Sequence ; Base Sequence ; DNA ; genetics ; DNA Restriction Enzymes ; genetics ; DNA-Directed DNA Polymerase ; genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; methods ; Plasmids ; Receptors, Polymeric Immunoglobulin ; genetics