Integrated e-clinical solutions in clinical research / 药学学报

Implementation of information technology in clinical research has resulted in revolutionary changes in drug development. Based on the good clinical practice (GCP) requirements on data, processes and documentations, and the era of fast growth in clinical studies using up-to-date information technology, we explore an integrated e-clinical solution in clinical studies in China.

Utility of high throughput sequencing technology in analyzing the terminal sequence of caudovirales bacteriophage genome / 病毒学报

To confirm the hypothesis that the high frequency sequences of high throughput sequencing are the terminal sequences of the bacteriophage genome. An adaptor of specific sequence was linked to the end of the bacteriophage T3 genomic DNA, which was then subject to high throughput sequencing; as a control, the same T3 genomic DNA without adaptor was also analyzed by high throughput sequencing. The sequencing results were examined with bioinformatics software. Similar high throughput sequencing technique was applied to analyze the genomic sequence of N4-like bacteriophage IME11. Bioinformatics study showed that the sequences tagged with adaptors were consistent with the high frequency sequences without adaptor labeling. Our analysis also indicated that the end of the T4-like phage genome had specific sequences instead of random sequences, disagreeing with the previous assertion. Evidences were provided that N4-like bacteriophage had a particular terminal sequence: the left end of the genome was unique while the right end was permuted. The high throughput sequencing technique was convenient and practical to be used to simultaneously detect the terminal sequence and the complete sequence of bacteriophage genome.

Construction of HCV-producing cell model based on self-cleaving ribozyme / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To construct a stable HCV-producing cell model for anti-HCV drug research.</p><p><b>METHODS</b>The HCV-ribozyme recombinant plasmid pJFH1-Rbz was constructed to generate the exact 5' and 3' ends of HCV genomic RNA by placing two self-cleaving ribozymes at both ends of the HCV JFH-1 cDNA. The plasmid was then transfected into HepG2 cells and the resultant clones were screened with G418. Subsequently, immunofluorescence and Western blot were performed to detect the expression of HCV core protein, HCV RNA level was quantitated by TaqMan real-time PCR method and HCV particles was detected by electron microscopy.</p><p><b>RESULTS</b>HCV core protein was detected in the screened cell clone, and the level of HCV RNA was up to 1000,0000 copies/ml in the culture medium. Electron microscopy showed the viral particles in the culture suspension were approximately 55 nm in diameter. IFN-treating experiment demonstrated that the HCV RNA level decreased with the increasing concentration of IFN alpha.</p><p><b>CONCLUSION</b>We constructed a stable HCV-producing cell model which can be used for anti-HCV drug research.</p>

Analysis of clinical and pathological features of chronic hepatitis B with hepatic steatosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore clinical and pathological features of chronic hepatitis B (CHB) with hepatic steatosis.</p><p><b>METHODS</b>Retrospective analysis of hepatic steatosis in patients with liver biopsy-proven CHB between January 2005 and June 2008. Detailed clinical, laboratory and pathological data of CHB patients with steatosis were compared with those in sex-, age- matched CHB patients without steatosis. Patients co-infected hepatitis C virus or HIV or suffering from liver diseases of other causes were excluded.</p><p><b>RESULTS</b>Histological hepatic steatosis was found in 33.4% of the 1263 CHB patients. The prevalence of steatosis was increased with time in the study period (20.3%, 28.2%, 32.6%, 65.4%, in trend analysis, P values less than 0.05). Body mass index, fasting plasma glucose, serum triglyceride and total cholesterol level in CHB patients with hepatic steatosis (n = 114) were significantly higher than those in 113 patients without steatosis (t values were 6.811, 2.733, 3.063, 2.340, respectively, P values less than 0.01 or 0.05). Compared to patients without steatosis, serum hepatitis B virus DNA titer in patients with steatosis was significantly lower (x2 = 6.154, P less than 0.05) and reduced sharply with the increased degree of hepatic steatosis (x2 = 4.941, P less than 0.05). There were no differences in liver biochemical test (t values were 0.744, 1.390, -0.029, -1.175, 1.393, respectively, P values more than 0.05), hepatic inflammation grade and fibrosis stage between CHB patients with and without steatosis (x2 = 1.434, 0.106, respectively, P more than 0.05), and these parameters were not associated with different degree of hepatic steatosis (x2 = 2.447, 2.911, respectively, P more than 0.05).</p><p><b>CONCLUSIONS</b>Hepatic steatosis is common in patients with CHB, and is related to metabolic disorders. Hepatic steatosis does not affect the severity of CHB. The reverse association of hepatitis B virus titer with the degree of hepatic steatosis needs further investigation.</p>

In Vitro Generation of Hematopoietic Stem Cells from Embryonic Stem Cell to Reconstruct Hematopoiesis / 实用儿科临床杂志

Objective To search for a good method for generation of hematopoietic stem cells (HSC) by embryonic stem cells (ESC),and to investigate the potential of HSC derived from ESC to reconstruct hematopoiesis in vivo.Methods Using a three-step method to induce a mice ESC line, E14.1-into HSC.And identifying HSC by flow cytometry analysis cell markers with CD34 +/Sca-1+, then HSC (1×109L-1)from ESC differentiation were injected into severe combined immunodefieency (SCID) mice for observing teratoma formation.To validate function of HSC by colonngenic cells assay and to reconstitute the hematopoiesis in lethally irradiated mice.Results Combining to use more hematopoietic stimulating factor to availably promote the El4.1 cell into embryoid body (EBs) with abundant hematopoietic progenitors.EBs were induced after 14 day to fast differentiate for HSC with peak percentages of CD34+/Sca-1+ cells reached to (13.72±2.07)%.To harvested ceils from EBs by day 14 for second -step HSC differentiation, percent of CD34+/Sca-1+ cells rise to(24.62±2.50) % after day 16 induction.Cloning forming units (CFU) analysis showed that more Erythro -myeloid cloning generation was observed at this period.Cells obtained in the second step are subsequently plated on monolayer of mice bone marrow stromal cells, in the presence of TPO, FLt3 ligand and superoatant of mice fetal liver stromal cells, cultured for additional 15 days, followed fast expansion of CD34+/Sca-1+ to maximally (58.64±4.20 ) % with more CFU-E, CFU-GM and CFU-GEMM population.Wright-Giemsa stain showed that its had the character of hematopoietic progenitors.Cells from the third-step were injected into SCID mice, but no teratomas were recovered in 2 mices after 6 weeks.Positive selection of CD34+/Sca-1+ cells by magnetic sorting from the third - step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 days after transplantation, with 71.4% successful engraftment rate.And 3 recipients showed that the cell population of the peripheral blood leukocytes,red cells, hemoglobin approached normal index at 40 days after transplant, hut followed relative'slow renew in platelet count.Survival rate of transplant group is 43.0%, compared to 100% mortality in control mice.Karyotyping assays confirmed female mice with XY.Conclusions The results showed that the three-step differentiation and the culture conditions described here good support differentiation of ESC into HSC.HSC derived from ESC were safe without teratomas formation in body, and its can reconstruct hematopoiesis.

The correlation of HBeAg expression and HBV-DNA in serum or peripheral blood mononuclear cells in patients with chronic hepatitis B / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the relationship between HBeAg expression and HBV-DNA in serum and peripheral blood mononuclear cells (PBMCs).</p><p><b>METHODS</b>208 patients with chronic hepatitis B were included in this present study. HBV-DNA in the PBMCs were performed by polymerase chain reaction (PCR), with the serum HBV-DNA level being determined by the way of fluoresces quantities PCR (FQ-PCR). Meanwhile, HBV-GM was also detected via enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>There were 106 patients for positivity in the HBV-DNA level of PBMCs with 102 for negativity, in which the HBV-DNA high levels (HBV DNA load > or = 1.0E5) in serum were 91.5%, 45.1% (chi2=52.12, P>0.01) respectively, with 76.4% and 50.9% (chi2=21.55, P>0.01) for the positive percentage of HBeAg expression.</p><p><b>CONCLUSION</b>A significantly positive correlation was found between HBV-DNA in PBMCs and serum HBV-DNA along with the positive percentage of HBeAg, indicating that obvious PBMCs' increase infected by HBV in patients with positivity of HBeAg and high level of serum HBV-DNA.</p>

Relationship between HBV-DNA in peripheral blood mononuclear cells and syndrome types of TCM in chronic hepatitis B patients / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the relationship between TCM syndrome type and HBV-DNA in serum and peripheral blood mononuclear cells (PBMCs) in chronic hepatitis B (CHB) patients.</p><p><b>METHODS</b>The serum HBV markers,HBV-DNA levels in serum and PBMCs, were quantitatively detected in 220 CHB patients by PCR method, and TCM syndrome type of 205 patients were differentiated.</p><p><b>RESULTS</b>Arranged from low to high, the percentages of CHB patients with the serum HBV-DNA > or = 1.0 x l0(5) copy/mL (high viral loading) in the five syndrome types were as follows: damp-heat retention in middle-jiao syndrome (DHRS, 55.2%), blood stasis blocking collateral syndrome (BSBC), Gan-Shen yin deficiency syndrome (GSYS), Pi-Shen yang deficiency syndrome (PSDS) and Gan stagnation with Pi deficiency syndrome (GSPS, 82.5%), the difference was significant between DHRS and GSPS; those with HBV-DNA in PBMCs infection were: GSYS (27.3%), DHRS (34.3%), BSBC (53.1%) and GSPS (77.2%). The percentage in GSPS was the highest, which was significantly different to that in other syndromes.</p><p><b>CONCLUSION</b>Amount of serum HBV-DNA and PBMCs HBV-DNA infection has certain correlation with the TCM syndrome type of CHB. The highest percentage of patients with HBV-DNA > or = 1.0 x l0(5) copy/mL and PBMCs HBV-DNA infection presented in CHB patients of GSPS type. We should pay more attention to strengthen genuine qi to eliminate pathogenic factors in treatment of CHB.</p>

Expression and clinical significance of KiSS-1 and E-cadherin in gastric cardia carcinoma / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the expression and clinical significance of KiSS- 1 and E- cadherin in gastric cardia carcinoma and the correlation between the two proteins.</p><p><b>METHODS</b>The expression of KiSS- 1 and E- cadherin in 80 patients with gastric cardia carcinoma and 20 patients with normal gastric cardia epithelium was detected by immunohistochemical technique.</p><p><b>RESULTS</b>The expression of KiSS- 1 was negatively correlated with lymphatic metastasis and clinical stage (P < 0.05), but not correlated with the cancer differentiation (P < 0.05). The expression of E- cadherin was negatively correlated with lymphatic metastasis, clinical stage, and cancer differentiation (P < 0.05). Spearman test showed a positive correlation between KiSS- 1 and E- cadherin expression (r(s)=0.722, P < 0.05).</p><p><b>CONCLUSION</b>KiSS- 1 and E- cadherin may play important roles in inhibiting the invasion and metastasis of gastric cardia carcinoma.</p>

Human umbilical vein endothelial cells as a feeder layer promote the growth of embryonic stem cells / 基础医学与临床

Objective To investigate whether human umbilical vein endothelial cells as a feeder layer was capable of supporting the growth of embryonic stem cells in vitro.Methods Human umbilical vein endothelial cells were isolated and cultured and then prepared as feeder cells after 3 passages.Alkaline posphatase activity(AKP) staining,stem cell surface marker test and karyotypes were conducted in different periods of cell culture.The suspension of stem cells cultured after 20 passages on endothelial cells were inoculated to the legs of severe combined immunodeficiency(SCID) mice subcutabeously to test the teratoma formation.Results E14.1 embryonic stem cells retained good colonies when they were cultured on endothelial cells for 3 passages and 8 passages.In addition,they expressed SSEA-1,Oct-4,and a membrane alkaline phosphatase to a high extent at passages 3 and 8.Embryonic stem cells cultured for 15 passages stably retaind a normal karyotype.Embryonic stem cells cultured on endothelial cells for 20 passages were inoculated into the hind leg of SCID mouse,teratomas containing three embryonic layers were recovered six weeks later.Conclusion Human umbilical vein endothelial cells would support effectively embryonic stem cells expansion,and provide a clinically safe method to expand ES cells for futureclinical application.

Human Umbilical Venous Endothelial Cells as Feeder Layer to Support the Growth of Embryonic Stem Cells / 实用儿科临床杂志

Objective To explore whether human umbilical venous endothelial cells could be used as feeder layer to support the growth of embryonic stem cells (ESC) and keep ESC undifferentiated.Methods The venous vessels of umbilical cord obtained from healthy puerperal were perfused with collagenase.The isolated endothelial cells went through primary culture and passages for expansion.Factor Ⅷ antigens determination was implemented.Endothelial cells with good growth and 3 or above passages were treated with mitomycin-C(10 mg/L) and prepared as feeder layer,on which E14.1 ESC was transplanted for subculturing to observe the morphological characterization and determine ESC alkaline psphatase (AKP) activity and the expression of stem cell marker Oct-4.Severe combined immune deficiency(SCID) mouse in vivo terotoma formation experiment was performed to identify its pluripotent properties.Results Human umbilical vein-derived endothelial cells grew well in culturing in vitro and regenerate in large numbers.The endothelial cells maintained normal cellular morphological and biological characterization after 10 passages.The cells stopped proliferating after being treated with mitomycin-C,but its activity and morphological properties were well-maintained with 24 hours,which was a fundamental property of serving as feeder layer.E14.1 ESC remained undifferentiated in human umbilical venous endothelial cells after 3-8 passages,the cells grew in colony and showed high expression of AKP and stem cell Oct-4.In vivo pluripotency experiment showed that 6 weeks after being transplanted to SCID mice E14.1 ESC of 6 and 10 passages in endothelial cells both could form teratoma containing 3 layers of tissue cells.Conclusions Human umbilical venous endothelial cell serve as a convenient feeder layer cell with rich sources.It can effectively support ESC growth and heterogenous and prevent the heterogeneous protein pollution and pathogenic microorganisms caused by animal cell feeder layers,thus solve the problem of biological safety of ESC clinical application.

Induction of apoptosis in human hepatoma cell line SMMC7721 by Newcastle disease virus HN gene / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the mechanisms of apoptosis induced in human hepatoma cell line SMMC7721 by plasmid pVHN constructed with Newcastle disease virus (NDV) HN gene.</p><p><b>METHODS</b>Twenty-four h after transfection with liposome-plasmid pVHN complexes in vitro, the mortality rate of SMMC7721 cells was determined by MTT staining and flow cytometry (FCM) with PI staining. The alteration of mitochondrial trans-membrane potential of the cells was detected by FCM with rhodamine 123 staining. Cell genomic DNA was detected by agarose electrophoresis. The activation of caspase-3 was assayed by its substrate color reaction.</p><p><b>RESULTS</b>Significant apoptosis was induced by transfection with plasmid pVHN into the cells for 24 h and the mortality rate was 50.0% (the mortality rate of control group was 5.2%). Genomic DNA was fragmented and mitochondrial trans-membrane potential was decreased, but caspase-3 activity increased.</p><p><b>CONCLUSION</b>Significant apoptosis in SMMC7721 cells can be induced by NDV HN gene. Apoptosis may be resulted from the decrease of mitochondrial trans-membrane potential and activation of Caspase-3.</p>

Construction of the nucleic vaccine pVVP3L-18HN and its antitumor effect on human laryngeal carcinoma / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>Nucleic vaccine of pVVP3IL-18HN expressing apoptin gene, Newcastle disease virus HN gene and IL-18 gene were constructed to observe the combinative antitumor effect of the above three genes.</p><p><b>METHODS</b>Eukaryotic expression plasmid pVVP3IL-18HN was constructed by inserting apoptin gene and fragment comprising fused IL-18HN gene and IRES promoter into the downstream of CMV promoter of vector pVAX1. The expression of inserted gene was identified by RT-PCR, indirect immunofluorescence and Western-blot. The recombinant plasmid was introduced into Hep-2 cells by liposome, then suppression rate of Hep-2 of different time and different quantity was calculated according to MTT results.</p><p><b>RESULT</b>The recombinant plasmid of pVVP3IL-18HN suppressed Hep-2 successfully and its suppression rate was up to 61.9% with 20 microg/ml, incubation of 72 hours.</p><p><b>CONCLUSION</b>The nucleic vaccine constructed pVVP3IL-18HN had antitumor effect on Hep-2. It may can be used to the therapy and research of laryngeal carcinoma.</p>

In vitro hematopoietic stem cells from embryonic stem cells reconstruct hematopoiesis in mice / 中国病理生理杂志

AIM:To investigate the potential of hematopoietic stem cells(HSCs)derived from mouse embryonic stem cells(ESCs)to reconstruct hematopoiesis in vivo.METHODS:Using a three-step method,a mice embryonic stem cell line,E14.1 was induced into hematopoietic stem cells.The cell markers with CD34+/Sca-1+ were identified by flow cytometry analysis,then HSCs(1?109 cells/L)from third-step were injected into SCID mice for observing teratoma formation.To validate function of HSCs,colonogenic cell assay was conducted and the hematopoiesis in lethally irradiated mice was reconstituted.RESULTS:The method of three-step differentiation,combined to use more hematopoietic stimulating factor promoted the E14.1 cell differentiation into HSCs with highest percent of CD34+/Sca-1+ cells(as high as 58.64%?4.20%)with more CFU-E,CFU-GM and CFU-GEMM populations.The cells showed the character of hematopoietic progenitors by Wright-Giemsa staining.Positive selected CD34+/Sca-1+ cells by magnetic sorting from third-step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 d after transplantation,with 71.4%(5/7)successful engraftment rate.Three recipients showed that the cell population of the peripheral blood leukocytes,red blood cells and hemoglobin approached to normal index at 40 d after transplantation,but followed relative slow renew in platelet count.Survival rate in transplant group was 43%,compared to 100% mortality in control mice.Karyotyping assays confirmed the female mice with XY.CONCLUSION:The three-step differentiation and the culture conditions described here support the differentiation of mouse ESCs into HSCs.HSCs derived from mouse ESCs can reconstruct hematopoiesis.