[Effects of HOXB4-transfecting directly and HOXB4-transfected HUCMSC on in vitro expansion of human umbilical cord blood CD34+ cells].

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.

Significance of TFF3 protein and Her-2/neu status in patients with gastric adenocarcinoma.

Increased knowledge of molecular alterations involved in gastric carcinogenesis has provided useful information for diagnosis and prediction. In this study, we investigated the clinicopathological significance of TFF3 expression and Her-2/neu status in gastric adenocarcinoma and explored the correlation between TFF3 expression and Her-2/neu status. A hundred and twenty-six (126) patients having undergone curative gastrectomy were enrolled. Immunohistochemistry for TFF3 was performed on tumor tissues and non-neoplastic resection margins. Her-2/neu status was assessed by immunohistochemical staining and fluorescence in situ hybridization (FISH) on tumor tissues. As a result, TFF3 expression and Her-2/neu positivity were detected in 46.8% and 11.9% of the cases, respectively. Patients with negative TFF3 exhibited longer survival than the positive group (P=0.0142), while patients with positive Her-2/neu only showed a tendency toward longer overall survival (P>0.05). However, Her-2/neu was significantly associated with improved disease-free survival (P=0.0234). Multivariate analysis demonstrated Her-2/neu and TFF3 to be independent prognostic indicators of recurrence, and a significantly poor prognosis for expression of TFF3 in patients with Her-2/neu negative tumors. TFF3 is an independent indicator for overall survival in gastric cancer, while Her-2/neu, as a marker of long time survival prediction, seems to be limited.

[Expression and significance of vitamin D and its receptor mRNA in the peripheral blood of initial immune thrombocytopenic patients].

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.

Construction and expression of an eukaryotic expression vector containing the IER3 gene.

BACKGROUND: More and more research indicate that the immediately early response gene 3 (IER3) is involved in many biological processes, such as apoptosis and immunoreaction, as well as viral infection, tumorigenesis and tumour progression. METHODS: Here we describe the construction of an eukaryotic expression vector containing IER3 gene and its expression in A549 cells as assessed through fluorescence microscopyand Western- blotting. RESULTS: Fluorescence detection displayed that GFP in cytoplasm was high during 48 and 72 hours post-transfection. In addition, Western blotting showed significant increase in IER3 gene expression in the transfected cells compared with controls. CONCLUSION: The recombinate plasmid expression vector was constructed successfully, which may provide a basis for further exploration of function of IER3 in lung cancer.

[Correlation of angiopoietin-2 and angiopoietin-2 receptor expressions in serum and placenta with preeclampsia].

OBJECTIVE: To investigate the correlation of the expressions of angiopoietin-2 (Ang-2) and angiopoietin-2 receptor (Tie-2) in serum and placenta with preeclampsia. METHODS: From May 2009 to April 2010, 62 women with preeclampsia who delivered in Affiliated Hospital of Qingdao University Medical College were recruited in the study, including 30 women with moderate preeclampsia (MPE group) and 32 women with severe preeclampsia (SPE group). Another 30 healthy pregnant women were taken as control group. ELISA was used to measure the serum Ang-2 in these women. Semiquantitative reverse transcription (RT)-PCR was used to investigate the expressions of Ang-2 mRNA and Tie-2 mRNA in placenta. Western blot was used to determine the expression of Ang-2 protein in placenta. RESULTS: (1) The serum concentrations of Ang-2 in MPE group and SPE group were (5.4 ± 1.8) µg/L and (5.1 ± 1.7) µg/L, respectively. Both were significantly lower than that in control group (16.2 ± 4.5) µg/L (P < 0.01). There was no significant difference between MPE group and SPE group (P > 0.05). (2) The expressions of Ang-2 mRNA in placenta of MPE group (2.1 ± 0.7) and SPE group (2.0 ± 0.6) were both significantly lower than that of control group (5.8 ± 0.8; P < 0.01). But there was no significant difference in Ang-2 mRNA expression between MPE group and SPE group (P > 0.05). (3) No significant difference was found in the expressions of Tie-2 mRNA in placenta among MPE group (1.33 ± 0.04), SPE group (1.35 ± 0.05) and control group (1.34 ± 0.04; P > 0.05). (4) The expressions of Ang-2 protein in placenta of MPE group (2.0 ± 0.8) and SPE group (2.0 ± 0.8) were both significantly lower than that of control group (5.7 ± 0.9; P < 0.01), while no significant difference was found between MPE group and SPE group (P > 0.05). (5) In MPE group and SPE group, the serum concentrations of Ang-2 were positively correlated with the levels of Ang-2 mRNA and Ang-2 protein in placenta (r = 0.651, 0.627; P < 0.01). CONCLUSIONS: Decreased expressions of Ang-2 mRNA and Ang-2 protein in placenta reduced serum concentration of Ang-2. Low expression of Ang-2 may be involved in the pathophysiological process of preeclampsia by affecting the formation of placenta in early pregnancy.

[Relationship between variation of coxsackievirus B3 VP1 sequence from cerebrospinal fluid of children and severity of damage to central nervous system].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Expression of RhoA and RhoC in colorectal carcinoma and its relations with clinicopathological parameters.

BACKGROUND: Ras homologous (Rho) family GTPases play a pivotal role in the regulation of numerous cellular functions associated with malignant transformation and metastasis. To evaluate the role of these GTPases in colorectal cancer, the mRNA expression levels in matched sets of tumor and non-tumor tissues from surgical specimens were analyzed. The relationship between the mRNA levels in tumor tissues to the clinicopathological features was also assessed. METHODS: A total of 68 patients with colorectal carcinoma were recruited and the levels of RhoA and RhoC mRNA transcripts in cancer, paratumoral and normal tissues were characterized by quantitative real-time polymerase chain reaction (QRT-PCR). Their correlation to clinical histopathological parameters was analyzed. RESULTS: The levels of RhoA and RhoC mRNA transcripts in carcinoma tissues were significantly higher than those in the matched paratumor and normal tissues from the same patient (p<0.05). The expression levels of both genes were significantly correlated with metastasis of cancer cells to lymph nodes and liver (p<0.05). The levels of RhoA expression were significantly correlated with the histopathological degree of cancer, while the expression of RhoC was correlated with the extent of local invasion to intestine. CONCLUSIONS: This is the first study with QRT-PCR to examine the expressions of RhoA and RhoC genes in colorectal carcinoma of Chinese patients. The significantly up-regulated RhoA and RhoC expressions suggest that they may contribute to the initiation, development, invasion and metastasis of colorectal carcinoma in Chinese patients.

Inhibitory effects of adenovirus mediated tandem expression of RhoA and RhoC shRNAs in HCT116 cells.

BACKGROUND: RhoA and RhoC are deregulated by over expression in many human tumors, including colorectal cancer. Some reports show that they play a pivotal role in the carcinogenesis, tumor development and infiltration metastasis. In this study, for the first time we constructed recombinant adenovirus to investigate the inhibitory effects of RhoA and RhoC shRNAs in tandem expression on the cell proliferation and invasion of colorectal cancer HCT116 cells. METHODS: The recombinant adenovirus carrying RhoA and RhoC shRNAs in tandem expression was transfected into HCT116. The mRNA transcription and protein expressions of RhoA and RhoC were examined by RT-FQPCR and Western blot respectively. Cellular proliferation inhibitory activity was determined by methyl thiazolyl tetrazolium (MTT) assay and invasive and migrating potential was detected through in vitro Matrigel coated invasion and migration assay. RESULTS: Both mRNA and proteins Levels of RhoA and RhoC were significantly reduced in HCT116 cells transfected with Ad-A1+A2+C1+C2 than those in Ad-HK group and control one. The relative RhoA and RhoC mRNA transcriptions were decreased to 40% and 36% (P < 0.05), while proteins expression reducing 42% and 35%, respectively (P < 0.05). Growth curves analysis showed that alive cell number in the Ad-A1+A2+C1+C2 group was lower than others in the third to sixth day and transwell chamber analysis showed that migration/invasion activity was significantly suppressed in Ad-A1+A2+C1+C2 group. CONCLUSION: Our results indicate recombinant adenovirus carrying RhoA and RhoC shRNAs in tandem expression may inhibit the growth and invasion of HCT116 cells. Application of such vector to inhibit one or more genes may be a new method to cancer therapy.

[Infection status and clinical significance of Epstein-Barr virus in pediatric leukemia---a report of 35 cases].

BACKGROUND & OBJECTIVE: Epstein-Barr virus (EBV) is associated with genesis of many human tumors. This study was to detect EBV infection in pediatric leukemia, and to explore its clinical significance. METHODS: EBV DNA in peripheral blood mononuclear cells in 35 pediatric leukemia patients, including 26 cases of acute lymphoblastic leukemia (ALL) (24 received initial treatment and 2 received retreatment), 8 cases of acute non-lymphocytic leukemia (ANLL) and 1 case of chronic lymphocytic leukemia (CLL), and in 14 healthy children was detected by fluorescent quantitative polymerase chain reaction (FQ-PCR). Its clinical significance was analyzed according to the clinical manifestations, prednisone sensitivity test, and complete remission (CR) rate after induction chemotherapy. RESULTS: EBV DNA was detected in 8 (22.86%) of the 35 pediatric leukemia patients. The positive rate of EBV DNA was 26.92% (7/26) in ALL with quantity of (5.144+/-6.91)x10(5) copies/ml, and 12.5% (1/8) in ANLL patients with quantity of 4.031x10(3) copies/ml. No EBV DNA was detected in CLL patients and healthy controls. The occurrence rates of peripheral leukocytosis and hepatosplenomegaly were significantly higher in the patients with EBV infection than in the patients without EBV infection (P <0.001). In ALL, the rate of no response to prednisone was significantly higher in the patients with EBV infection than in the patients without EBV infection (100% vs. 26.32%, P =0.001); CR rate after induction chemotherapy was significantly lower in the patients with EBV infection than in the patients without EBV infection (28.57% vs. 84.21%, P=0.003). In ANLL, the differences of CR rate and relapse rate were not significant between the patients with and without EBV infection (P=0.5). CONCLUSIONS: Pediatric leukemia patients with EBV infection have higher incidence of peripheral leukocytosis and hepatosplenomegaly. ALL patients with EBV infection have poor prednisone response and low CR rate.