Effects of HOXB4-transfecting directly and HOXB4-transfected HUCMSC on in vitro expansion of human umbilical cord blood CD34+ cells / 中国实验血液学杂志

This study was purposed to investigate the difference of nucleated cell (NC) count, CD34(+) cell ratio and expansion multiple, cell cycle and colony formation capability in in vitro expanded human umbilical cord blood CD34(+) cells from HOXB4-transfecting directly and HOXB4-transfected human umbilical cord mesenchymal stem cells (HUCMSC) by means of prepared feeder layers of HUCMSC. The HUCMSC were divided into 2 groups:first group, in which HOXB4 gene was transfected into HUCMSC by using lentiviral vecfor, and feeder layers were set up; and second group in which feeder layers for HUCMSC of non-transfected HOXB4 gene were set up. The CD34(+) cells were separated from HUCB by magmatic activated cell sorting(MACS). After culture in medium with cytokines for 2 days, CD34(+) cells were divided into 5 groups, including control group and experimental group. The control groups included CD34(+) cells as group A (blank control group) and GFP-CD34(+) cells as group B (negative control group) and experimental groups included HOXB4-CD34(+) cells as group C, HUCMSC+CD34(+) cells as group D, HOXB4-HUCMSC+ CD34(+) cells as group E and cells in all groups were cultured in vitro. The number of nucleated cells were counted at day 6, 10, 14 of culture and CD34 immunophenotypes, cell cycle and colony forming capability were measured at day 10 of culture in different conditions. The results indicated that HOXB4 gene could be transfected into HUCMSC by lentiviral vector and feeder layers were set up successfully. After culture for 14 days, the nucleated cells in 5 groups could be amplified effectively, and the expansion levels in 5 groups were in order HOXB4-HUCMSC+CD34(+) cell group> HOXB4-CD34(+) cell group>HUCMSC+CD34(+) cell group> control groups (P < 0.05). At day 10 of in vitro expansion the CD34(+) cell percentage decreased significantly in all groups, while the number of CD34(+) cell increased in experiment groups, which were in order HOXB4-CD34(+) cells group> HOXB4-HUCMSC+CD34(+) cell group>HUCMSC+CD34(+) cell group>control groups (P < 0.05). The cell cycle detection showed that the percentage of cells in S+G2/M phase in experiment groups were higher than that in control groups (P < 0.05), and percentage of cells in HOXB4-HUCMSC+CD34(+) cells group was higher (41.57%) than that in HOXB4-CD34(+) cells group(37.87%) and HUCMSC+CD34(+) cell group (28.65%) (P < 0.05). There was no statistical difference in the CFU number between HOXB4-HUCMSC+CD34(+) cell group and HOXB4-CD34(+) cell group, which were both higher than that in HUCMSC+CD34(+) cell group and control groups (P < 0.05).It is concluded that the CD34(+) cells cultured on HOXB4-HUCMSC feeder layers can be amplified significantly and kept the characteristics of stem cells, The feeder lager of HOXB4-HUCMSC is relative safe for amplification of CD34(+) cells in vitro, it possesses the potential useful value.

Expression and significance of vitamin D and its receptor mRNA in the peripheral blood of initial immune thrombocytopenic patients / 中国实验血液学杂志

To investigate the effect of vitamin D in pathogenesis and clinical treatment of patients with immune thrombocytopenic (ITP), ELISA was used to detect the level of 25-hydroxylate vitamin D3[25(OH)D3] and 1,25-dihydroxyvitamin D3[1,25(OH)2D3] in peripheral blood from 45 ITP patients and 30 normal controls. Vitamin D receptor (VDR) mRNA expression was detected by RT-PCR. The results showed that the levels of 25(OH)D3 (10.6 ± 7.7 ng/ml) and 1,25(OH)2D3 (69.9 ± 29.0 pg/ml) in peripheral blood of the initial ITP patients were lower than those in peripheral blood of normal controls (13.7 ± 9.1 ng/ml, 87.3 ± 19.9 pg/ml) (P < 0.05). The expression of VDR gene obviously increased in peripheral blood of initial ITP patients (1.588 ± 0.127), as compared with that in peripheral blood of normal controls (1.055 ± 0.734) (P < 0.05). It is concluded that vitamin D level and its receptor expression may play an important role in ITP, and vitamin D and its similarities may be a new agent to treat patients with ITP.

Dynamic change of collagen typeⅠand fibronectin in posterior sclera of form-deprivation myopia after TIMP-2 gene transfect in guinea pig / 中华实验眼科杂志

Background The domestic and international researches discovered that many proteins and enzymes of the extracellular matrix (ECM) participate in the sclera remodeling by affecting the collagen typeⅠand fibronectin.Objective This study was to investigate the effect of matrixmetalloproteinase-2 (TIMP-2) on expression of collagen typeⅠand fibronectin of ECM in the posterior sclera by injecting liposomes containing tissue inhibitor of TIMP-2 gene into suprachoroidal space of the form-deprivation myopia in guinea pig.Methods Form-deprivation myopia was induced by translucent goggles in 36 clean guinea pig for 2 weeks.Then the animals were randomly assigned to TIMP-2 group,empty plasmid group,saline group and 12 for each group.Liposomes of 5μl containing TIMP-2 gene,empty plasmid and saline were suprachoroidally injected in the right eye respectively,and the left eyes without any treatment were used as self-control group.Other 12 matched guinea pigs only covered the right eyes through out the experimental duration as model control group.The guinea pigs were sacrificed and the posterior sclera tissue of the eyeballs were collected at 2,7 and 14 days after injection of drug.The expressions of collagen typeⅠmRNA and fibronectin mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).This study followed the Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results The expression level of collagen type Ⅰ mRNA in the posterior sclera of guinea pig was lower but that of fibronectin mRNA was higher in TIMP-2 group than self-control group,showing significant differences between them (P＜0.05).The expression level of collagen type Ⅰ mRNA in the posterior scleral tissue began to increase from the 2nd day after drug injection and was obviously elevated at the 7th day and then gradually decreased at the 14th day.However,the expression level of fibronectin mRNA in the posterior scleral tissue showed the opposite pattern.The expression levels of collagen typeⅠmRNA and fibronectin mRNA at the 7th after drug injection were significantly lower than that at the 2nd day or 14th day (P＜0.01).Conclusion Suprachoroidal injection of TIMP-2 in form-deprivation myopia could up-regulate the expression of collagen typeⅠmRNA and down-regulate the expression of fibronectin mRNA in the posterior scleral tissue.It may slow down the sclera remodeling of form-deprivation myopia in guinea pig in the early stage.

Relationship between variation of coxsackievirus B3 VP1 sequence from cerebrospinal fluid of children and severity of damage to central nervous system / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the possible relationship between variation of coxsackievirus B3 (CoxB3) VP1 sequence from cerebrospinal fluid of children with severe and mild central nervous system (CNS) infection and damage to CNS in children from Shandong province.</p><p><b>METHODS</b>The enteroviruses were detected using VP1 typing and sequencing primer for enteroviruses from 73 enterovirus-infected cases confirmed by detection of cerebrospinal fluid by enteroviruses common primer. VP1 sequences (450 nucleotides) were determined and analyzed for 21 CoxB3 enteroviruses strains isolated in Qingdao and Binzhou, and were compared with that of BLAST search procedures from GeneBank in NCBI. The variation of VP1 gene and amino acids sequence of CoxB3 enteroviruses was analyzed for severe and mild CNS infection.</p><p><b>RESULTS</b>The nucleotide homogeneity of these CoxB3 appeared to be 97% - 99%, however, the homogeneity among different genotypes were 83% - 76%. Replacement of glutamine by histidine at amino acid locus 856 of VP1 CoxB3 was found in 4 cases with severe encephalitis. There were different variation in VP1 nucleotide sequence of CoxB3 in 3 cases with mild encephalitis and 14 cases with meningitis, but amino acids sequences had no regular variation. The modified Glasgow's coma score was below 7 in all the 4 cases with severe encephalitis. Of these 4 cases, 3 had consciousness disturbance for less than 3 days. Lethargy, restlessness and psychiatric symptoms were major manifestations, of whom 3 also had dysphagia, 1 had encephalatrophy obviously, Glasgow's coma score was 3, deep coma lasted for 9 days, and had concomitant fatal epileptic attacks. Of these 4 cases, 2 completely recovered, 1 had high muscle tone, 1 remained under anti-epileptic drug treatment at follow-up 6 months later.</p><p><b>CONCLUSION</b>There were a small epidemic of CoxB3 CNS infection in children in 2005 in this area. The amino acid variation of CoxB3 VP1 possibly caused increased viral virulence and caused damage to CNS.</p>

Expression of Fas/FasL and the apoptosis of HepG2 cells transfected with LIGHT and IFN-? / 中国现代普通外科进展

Objective:To investigate the expression of Fas、FasL and the apoptosis of liver cancer cell line HepG2 transfected with LIGHT and IFN-? gene mediated by Cationic liposome.Methods:HepG2 cells were divided into two groups(the solo transfection of LIGHT gene and the combined transfection of LIGHT and IFN-? genes) and the control groups(no transfection).HepG2 cells were cellected at 12h,24h and 48h after transfection.The apoptosis of HepG2 cells and the expression of Fas and FasL of the HepG2 cells were investigated with flow cytometry.Results:After transfection,the apoptosis of HepG2 cells increased,and the apoptosis of combined transfection group was higher than the solo transfection of LIGHT(P