Effects of regenerated tissue extracts after liver injury on the proliferation, differentiation, migration and invasion of SK-HEP1 cells

Objective: To study the effects of regenerated tissue extracts after liver injury on the proliferation, differentiation, migration and invasion of SK-HEP1 cells. Methods: Regenerated tissue extracts after liver injury were used to induce SK-HEP1 cells after enrichment, their effects on the proliferation, differentiation, migration and invasion of SK-HEP1 cells were observed through in vitro cell culture, MTT, flow cytometry and transwell assays. Results: In response to the action of regenerated tissue extracts after liver injury, SK-HEP1 cells were blocked in G

Effects of regenerated tissue extracts after liver injury on the proliferation, differentiation, migration and invasion of SK-HEP1 cells

Objective:To study the effects of regenerated tissue extracts after liver injury on the proliferation, differentiation, migration and invasion of SK-HEP1 cells.Methods:Regenerated tissue extracts after liver injury were used to induce SK-HEP1 cells after enrichment, their effects on the proliferation, differentiation, migration and invasion of SK-HEP1 cells were observed through in vitro cell culture, MTT, flow cytometry and transwell assays.Results:In response to the action of regenerated tissue extracts after liver injury, SK-HEP1 cells were blocked in GConclusions:To a certain extent, regenerated tissue extracts after liver injury can inhibit the proliferation, differentiation, migration and invasion of hepatoma cells, showing an important potential of being a differentiating agent for the treatment of liver cancer.

Construction and identification of the recombinant adenovirus vector carrying a small interfering RNA targeting the peroxisome proliferator-activated receptor-γ / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Steroid-induced osteonecrosis of the femoral head (ONFH) is a common clinical disease, with a high disability rate. At present, efficient prevention and treatment of steroid-induced ONFH is still lacking. The peroxisome proliferator-activated receptor-γ (PPARγ) is recognized as an important pathogenic gene for the development of steroid-induced ONFH. RNA interference (RNAi) is a tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target proteins. Therefore, down-regulation of PPARγ expression by RNAi may prevent the incidence of steroid-induced ONFH.</p><p><b>METHODS</b>According to the principles of siRNA design, three duplex siRNA sequences (971 - 989, 1253 - 1271 and 1367 - 1385) derived from the PPARγ gene (NM_001082148) were synthesized. These duplexes were annealed, purified and ligated into 1.0-cytomegalovirus (CMV) shuttle vector. The shuttle vector was transfected into HEK293 cells. The HEK293 generated recombinant adenovirus vector carrying PPARγ siRNA sequences was purified and the titer of recombinant adenovirus was determined.</p><p><b>RESULTS</b>After the annealing of single-strand DNA oligo encoding short hairpin RNA (shRNA) sequences, products were identified by gel electrophoresis. These products were ligated into the 1.0-CMV shuttle vector and the recombinant shuttle vectors 1.0-CMV-971, 1.0-CMV-1253 and 1.0-CMV-1367 were constructed. These sequences of these recombinant vectors were confirmed. We then successfully constructed the recombinant adenovirus vector carrying siRNA targeting PPARγ. After purification, the virus titer was higher than 10(10) plaque forming unit (PFU)/ml.</p><p><b>CONCLUSION</b>In this study, three recombinant adenovirus shuttle vectors carrying siRNA targeting PPARγ, including shuttle vectors 1.0-CMV-971, 1.0-CMV-1253 and 1.0-CMV-1367, were successfully constructed and high titers of recombinant adenovirus were obtained.</p>

Osteogenic potential of human calcitonin gene-related peptide alpha gene-modified bone marrow mesenchymal stem cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Most of the basic and clinical studies of osteonecrosis of the femoral head (ONFH) are restricted to bone tissues only, whereas various systems are involved in the onset and development of ONFH, including nervous system. Peptidergic nerve participates in the neuronal regulation of bone metabolism and anabolism, and plays key roles in the growth, repair and reconstruction of bone. Calcitonin gene-related peptide (CGRP), which is secreted by peptidergic nerve, is the main mediator of bone metabolism. It dramatically promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Additionally, it enhances the osteoblast mass and the rate of osteoblast formation, and reduces the bone resorption by acting on osteoblasts and osteoclasts. Hence, we aimed to construct recombinant retrovirus vector pLNCX(2)-hCGRPα and to investigate the proliferation and osteogenic potential of hCGRPα-producing BMSCs (BMSCs/pLNCX(2)-hCGRPα) after virus infection.</p><p><b>METHODS</b>The constructed recombinant retrovirus vector pLNCX(2)-hCGRPα was transfected into PT67 packaging cells by lipofectamine 2000. Virus was collected for BMSCs infection. The mRNA and protein expression of hCGRPα was examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. The cell proliferation was determined by methyl thiazoleterazolium (MTT) assay. The osteogenic potential of BMSCs was evaluated by alkaline phosphatase (ALP) activity.</p><p><b>RESULTS</b>Both mRNA and protein expression of hCGRPα was detected in BMSCs/pLNCX(2)-hCGRPα cells. These cells exhibited significantly elevated proliferation and ALP value as compared with control BMSCs (P < 0.05).</p><p><b>CONCLUSION</b>BMSCs/pLNCX(2)-hCGRPα cells could stably express hCGRPα and showed promoted proliferation ability and osteogenic potential as compared with control BMSCs.</p>

Effect of CEA gene regulation on the anti-tumor activity of oncolytic adenovirus H101 to esophageal carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the effect of CEA gene regulation on the anti-tumor activity of oncolytic adenovirus H101 to esophageal carcinoma, and to explore the intrinsic factors influencing H101 sensitivity.</p><p><b>METHODS</b>Stable human esophageal cancer cell line EC9706 cells with lower (EC9706-SCEA) and higher CEA expression (EC9706-CEA) were chosen, thawed and cultured, and then to analyse the influence of CEA expressed at different levels on cell growth. The cytotoxic effect of H101 was assayed by in vitro and nude mouse in vivo.</p><p><b>RESULTS</b>The cell growth experiment showed that the population doubling time of EC9706-SCEA, EC9706-CEA and EC9706 cells were (30.9 ± 2.0) h, (31.1 ± 2.5) h and (29.1 ± 2.6) h, respectively, showing no significant difference among them (P > 0.05). The cytotoxic activity of H101 was higher on EC9706-SCEA than on other four groups, when MOI was ≥ 0.01 PFU (P < 0.05). The mouse experiment showed that H101 inhibited the growth of transplanted tumors in all experimental groups. Its effect on CEA-silenced tumors (inhibition rate was 61.5% to 74.5%) was significantly higher than that on CEA-overexpression tumors (32.3% to 38.5%) and control EC9706 transplanted tumors (35.5% to 44.8%). There was a significant difference between them (P < 0.05).</p><p><b>CONCLUSIONS</b>The results in vitro and in vivo experiments show that H101 can enhance the cytotoxic effect on EC9706 cells with lower CEA expression. To silence the expression of CEA may provide a novel strategy for target gene therapy of esophageal carcinoma.</p>

In-vitro amplification of oval cells with preservation of stem cell phenotype / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore cell culture techniques for amplification of oval cells with preservation simultaneously of the stem cell characteristics.</p><p><b>METHODS</b>Oval cell line OC3 was cultured in RPMI 1640 supplemented with 15% fetal bovine serum and 20 µg/L EGF. Cells were harvested every 5 passages and were examined with biomarkers including OV-6, c-kit, gamma-glutamyl transpeptidase, placental form of glutathione-S-transferase (GST-P), pyruvate kinase M₂, pyruvate kinase L and albumin using techniques including RT-PCR, immunocytochemistry, and enzymo-cytochemistry.</p><p><b>RESULTS</b>OC3 cell lines could be amplified abundantly in-vitro associating with expression of infant liver cell markers at various level, including OV-6, c-kit, gamma-glutamyl transpeptidase, GST-P, pyruvate kinase M₂, but no expression of mature hepatocyte markers detected including pyruvate kinase L and albumin.</p><p><b>CONCLUSIONS</b>Amplification of OC3 cells with preservation of the stem cell phenotype and high proliferation index can be achieved up to the 79(th) passages by culturing in RPMI 1640 supplemented with 15% fetal bovine serum and 20 µg/L EGF.</p>

Construction of a recombinant lentiviral expression vector carrying carcinoembryonic antigen gene and its expression in dendritic cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a lentiviral expression vector of human carcinoembryonic antigen (CEA), and identify its expression in dendritic cells (DCs).</p><p><b>METHODS</b>Human CEA-encoding sequence was amplified, purified, ligated with lentiviral vector plasmid pLentiGFP and verified by sequencing. The verified recombinant vector plasmid (pLentiGFP-CEA), the packaging plasmid p 8.2 and pVSV-G were transfected into 293T cells by Lipofectamine(TM) 2000 reagent. The supernatant of the cultured 293T cells was collected to infect the DCs. The expression of CEA in the transfected DCs was assayed by RT-PCR and Western blotting.</p><p><b>RESULTS</b>CEA lentiviral vector was highly expressed in the transfected DCs as observed using fluorescence microscope 48 h after the the transfection. The human CEA gene was successfully amplified by RT-PCR with a length of about 2100 bp. Western blotting also showed CEA expression in the transfected DCs.</p><p><b>CONCLUSION</b>The human CEA lentiviral expression vector has been successfully constructed and the functional CEA protein can be expression in the transfected DCs. This facilitates further studies of the function of CEA at the molecular level.</p>

A cross-sectional study of congenital heart disease among children aged from 4 to 18 years at different altitudes in Qinghai province, China / 中华流行病学杂志

<p><b>OBJECTIVE</b>The epidemiological characteristics of congenital heart disease (CHD) in children aged from 4 to 18 years were investigated in Qinghai province.</p><p><b>METHODS</b>Altogether 288 066 children inhabiting at 6 prefectures and 3 counties were examined by the following three steps: pre-screening, re-examination and diagnosis with color Doppler. And the entity distribution was analyzed while the differences were compared by age, gender, altitudes and nationalities respectively.</p><p><b>RESULTS</b>Altogether 1633 cases of CHD were discovered. The total prevalence of CHD was 5.71 per thousand. The prevalence of CHD was found to increase with the increase of altitude by 4.89 per thousand at the altitude of 2535 m, 5.71 per thousand at 3600 m, and 8.74 per thousand at 4200 m respectively. There were significant differences among different altitude (chi(2) = 54.696, P < 0.001). chi(2) trend analysis showed the increase with chi(2) = 41.826(P < 0.001). The total incidence of CHD in females was 6.95 per thousand, which was significantly higher than that in males with 4.54 per thousand (chi(2) = 73.79, P < 0.001). There were significant differences between males and females at the altitude of 3000 m (chi(2) = 84.733, P < 0.001) and 4000 m (chi(2) = 16.313, P < 0.001) except at the altitude of 2000 m (chi(2) = 0.807, P > 0.05). The prevalence of CHD in different age groups was statistically significant at the every altitude of 2000 m (chi(2) = 18.138, P < 0.001), 3000 m (chi(2) = 18.544, P < 0.001) and 4000 m (chi(2) = 27.535 P < 0.001). The prevalence of CHD was increasing with the increase of age groups at the altitude of 3000 m (chi(2) = 19.230, P < 0.001) and 4000 m (chi(2) = 26.894, P < 0.001) except at the altitude of 2000 m. Within the prevalence of CHD of different nationalities, there was a significant difference with chi(2) = 24.456 (P < 0.001). Within the constituent rate of CHD, the prevalence of atrial septal defect (ASD) was as high as 37.42%, followed by the prevalence of patent ductus arteriosus (PDA) as 28.47% and ventricular septal defect (VSD) as 26.01%. Regarding the four categories of CHD, the constituent rate varied at different altitudes. For example, the prevalence rate of ASD constituted 37% at the altitude of 2000 m and 3000 m, and that of PDA accounted for 46.36% at the altitude of 4200 m.</p><p><b>CONCLUSION</b>The epidemiological characteristics of CHD in Qinghai children were possibly associated with altitude levels.</p>

Epidemiological survey of congenital heart disease among people aged from 4 to 18 in Haidong area of Qinghai province / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the epidemiological characteristics of congenital heart disease (COHD) among 4 to 17 years old children in Haidong area of Qinghai province.</p><p><b>METHODS</b>All 97 718 children were surveyed with the following 3 steps: prescreening, countershock and confirmation with color Doppler. The distribution patterns were analyzed by national groups, ages and genders respectively.</p><p><b>RESULTS</b>There were 496 COHD cases detected. The total incidence was 5.076 per thousand (496/97 718). The incidences of male and female were 5.046 per thousand (256/50 730) and 5.108 per thousand (240/46 988) (chi(2) = 0.018, P > 0.05). There was a significant difference between Pingan county and the others (chi(2) = 10.62, P < 0.01). The highest incidence was in Ledu (5.46 per thousand), the incidences of Huzhu and Pingan county were 5.45 per thousand and 3.64 per thousand respectively. There was no significant difference among different national groups (chi(2) = 0.33, P > 0.05). Among 496 COHD cases, the ratio of atrial septal defect (ASD), ventricular septal defect (VSD), patent ductus arteriosus (PDA) were 37.30%, 35.69% and 22.18% respectively.</p><p><b>CONCLUSION</b>Total incidence of COHD was 5.076 per thousand in Haidong area of Qinghai province. The incidence was not different in both genders and national groups. The constitution of COHD in different counties were different.</p>

Preliminary study of DNA polymerase beta gene silencing by small interfering RNA in human gastric cancer BGC-823 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the influence of DNA polymerase beta (polbeta) gene silencing by small interfering RNA on biological behavior of human gastric cancer cell line BGC-823.</p><p><b>METHODS</b>The siRNA eukaryotic expression vectors targeting polbeta gene were constructed and transfected into BGC-823 cells by liposome. Stable cell lines were screened with G418. The expression levels of polbeta mRNA and protein were detected by real time PCR and Western blot in the cells of each group. The proliferation of each group was detected by flow cytometry and tumorigenicity was determined in nude mice.</p><p><b>RESULTS</b>The siRNA expression vector targeting polbeta gene was successfully constructed. The expression levels of polbeta mRNA and protein were significantly reduced in the experimental group transfected with siRNA expression vectors targeting polbeta, and the silencing effect of pRNAT-U6.1-sipolbeta2 (suppression degree was 83%) was stronger than that of pRNAT-U6.1-sipolbeta1 (depression degree is 56%). Compared with irrelevant siRNA control group, empty vector control group and untransfected group, the ratio of G0/G1 cells was increased, proportion of S phase cells and cell proliferation were decreased in the experimental group 1 cells transfected with pRNAT-U6.1-sipolbeta1 (P < 0.05). On the contrary, the ratio of G1/G0 was decreased, proportion of S phase cells and cell proliferation was increased in the experimental group 2 cells transfected with pRNAT-U6.1-sipolbeta2 (P < 0.05).</p><p><b>CONCLUSION</b>The siRNA expression vectors targeting DNA polymerase beta gene can significantly inhibit the expression of polbeta mRNA. Neither high nor extremely low expression of polbeta is beneficial to maintain the cellular physiological functions. The expression of polbeta silenced to a proper level by siRNA may play an important role in inhibiting tumorigenesis.</p>

Study on the epidemiology of congenital heart disease in Tibetan ethnic children aged from 4 t0 18 living at different altitudes in Qinghai province / 中华流行病学杂志

Objective The epidemiological characteristics of congenital heart disease(CHD)among Tibetan children whose age ranged from 4 to 18 at different altitude were investigated in Qinghai province.Methods 32 578 Tibetan children living at 2535 m,3600 m and 4200 m were surveyed with the following 3 steps:prescreened,counterchecked and diagnosed with color Doppler.The entity distribution was then analysed and the age and gender were compared respectively.Resuits 235 CHD cases were identified.The total morbidity was 7.21‰.CHD morbidity was rising with the increase of altitude with 5.45‰ at 2535 m,6.80‰ at 3600 m and 9.79‰ at 4200 m respectively.There were significant static differences between 4200 m and the others with χ2=7.002(P＜0.01)to 2535 m and χ2=5.540(P＜0.05)to 3600 m.However,there was no statistical difference between 2535 m altitude and 3600 m altitude.The morbidity in different age had no statistical difference at 2535 m altitude but statistically increased with the increase of age at 3600 m and 4200 m.The total ratio of 16-18 age was significantly higher than other age periods with χ2=10.79(P＜0.005)to 4-7 age period and with χ2=5.60(P＜0.05)to 8-12 age period.The atrial septal defect(ASD)morbidity rates in three places was 39.1%followed by ventricular septal defect(VSD)with 32.8%and patent duetus arteriosus(PDA)with 24.7%.However,the constitute of CHD was different in different altitudes that VSD with 43.5%at 2535 m.ASD with 42.8%at 3600 m and PDA with 50.8%at 4200 m which was the highest morbidity.Conclusion Morbidity.constitutes and difference in gender and age were related to altitude.

Construction of a siRNA vector targeting human MTA1 gene and the gene-silencing effect / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct an expression vector of siRNA targeting human MTA1 gene and observe its gene-silencing effect in esophageal carcinoma cells.</p><p><b>METHODS</b>The siRNA sequences targeting MTA1 gene were designed and synthesized with two complementary oligonucleotide strands. The oligonucleotide strands were annealed and recombined into pRNAT-U6.2 vector, which was identified by sequencing following transformation and amplification. The siRNA expression vector pRNAT-U6.2-MTA1 was transfected into human esophageal carcinoma EC9706 cells via liposome. RT-PCR and Western blotting were used to detect expression levels of MTA1 mRNA and protein in the transfected EC9706 cells, respectively.</p><p><b>RESULTS</b>The double-stranded oligonucleotide fragments of the siRNA targeting MTA1 gene were cloned into pRNAT-U6.2 vector, which was validated by sequence analysis. RT-PCR and Western blotting indicated that MTA1 mRNA and protein expressions were significantly decreased in the transfected cells, especially in those transfected with the siRNA targeting the sequence of GACCCTGCTGGCAGATAAA (481-499), which induced almost complete silencing of MTA1 protein expression.</p><p><b>CONCLUSION</b>The siRNA expression vector pRNAT-U6.2-MTA1 for silencing MTA1 gene expression in the esophageal carcinoma cells has been successfully constructed, which may facilitate further study for decreasing the invasive and metastatic potentials of malignant tumors by MTA1 gene silencing.</p>

Construction and identification of a eukaryotic expression vector for the small interfering RNA targeting nucleostemin gene / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a eukaryotic expression vector for the small interfering RNA (siRNA) targeting nucleostemin (NS) gene.</p><p><b>METHODS</b>The siRNA targeting NS gene was designed according to the sequence of NS mRNA available in GenBank. Three siRNA sequences were obtained, and the corresponding cDNAs were synthesized and inserted into plasmid pRNAT-U6.1 for constructing the recombinant plasmids, which were transformed into E.coli DH5alpha strain. The plasmids, after identification by PCR and DNA sequencing, were transfected into EC9706 cell line via liposome, and the mRNA and protein expressions of NS gene in the cells were determined by RT-PCR and Western blotting, respectively.</p><p><b>RESULTS</b>Three recombinant plasmids were identified by PCR and sequence analysis, the results of which showed correct insertion of the designed sequences in the plasmids. RT-PCR and Western blotting showed substantially decreased mRNA and protein expressions of NS gene in the transfected cells.</p><p><b>CONCLUSION</b>The recombinant plasmid expressing the siRNA targeting NS gene has been successfully constructed, which provides the basis for studying RNA interference of the NS gene.</p>

Construction and identification of matrix metalloproteinase inhibitor-1 siRNA eukaryotic expression vectors / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct TIMP-1 siRNA eukaryotic expression vectors and evaluate their effect on TIMP-1 mRNA expression in hepatic stellate cells.</p><p><b>METHODS</b>The combinant lone DNA with cutting sites of BamH I and Xho I enzyme according to the sequences of 447-465, 552-540 TIMP-1 of rats and nonspecific sequence were selected and cloned to pGEM-T vector and sub-cloned to pRNAT-U6.2. They were then identified by double enzyme digestion analysis and DNA sequencing. Three plasmids were transfected into T6 separately through an oligofectamine package. TIMP-1 mRNA expression was evaluated by RT-PCR.</p><p><b>RESULTS</b>Targeting sequences of TIMP-1 siRNA eukaryotic expression vectors were correct. TIMP-1 mRNA expression was significantly reduced by transfecting them into the T6.</p><p><b>CONCLUSION</b>We successfully constructed two TIMP-1 siRNA eukaryotic expression vectors and the transfected cells can significantly suppress the TIMP-1 expression.</p>

Expression of Livin Gene and Its Isoforms in Children with Gliomas / 实用儿科临床杂志

Objective To study the expression of novel inhibitor of apoptosis protein(IAP) family member livin gene and its two isoforms(livin ? and livin ?) in brain tissue of children with gliomas.Methods Livin ? and Livin ? mRNA were detected by real time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR) in brain tissue of 30 children with gliomas and 12 healthy children.Result The positive rate of Livin mRNA expression was 83.3%(25/30 cases)in 30 cases of children with gliomas,the positive rate of Livin mRNA was only 8.3%(1/12 case) in normal brain tissue,there was significant difference in 2 groups(P

Expression of Inhibitor of Apoptosis Protein Livin in Children with Acute Lymphocytic Leukemia / 实用儿科临床杂志

Objective To study the expression of Livin,a novel inhibitor of apoptosis protein(IAP)family member in children with acute lymphocytic leukemia(ALL).Methods Livin protein of 40 cases myeloid tissue of children with ALL and 20 cases that of non-leukemia children were assayed by streptomycin avidin-biotin-peroxidase complex staining immunohistochemical method in order to analyze the relationship between Livin protein expression and development of ALL.Results The positive rates of Livin protein expression was 40% in 40 cases ALL,but in control group,the positive rates of Livin protein expression was 5%.The difference of 2 groups was significant(P