Genotyping of nucleocapsid protein gene of HCV in HIVHCV co-infected patients in Kunming in 2019 / 中国热带医学

@#Abstract: Objective　To investigate the distribution characteristics of HCV genotypes and subtypes in patients with HIV (human immunodeficiency virus, HIV)/HCV co-infection in Kunming based on the nucleocapsid protein gene sequence of HCV (hepatitis C virus). Methods　Serum was collected from HIV/HCV co-infected patients with household registration in 14 county-level cities, districts and counties under the jurisdiction of Kunming, who admitted to Yunnan Provincial Infectious Disease Hospital from March to August 2019. The viral RNA was extracted from the serum, reverse transcribed to synthesize cDNA, and the HCV nucleocapsid protein gene-specific primers were used for nested PCR amplification. The positive amplification products were sequenced, bioinformatics software such as DNAstar and MEGAX were used for sequence analysis. Results　A total of 64 samples from co-infected patients with clinical diagnosis of suspected HIV/HCV were collected and amplified by HCV nucleocapsid protein gene-specific primers, of which 17 samples were amplified positively. The results of sequence analysis showed that the sequences of 9 cases were located in the same evolutionary branch as the HCV 3b subtype sequence, and the nucleotide homology was 93.3%-95.2%; the sequences of 5 cases were located in the same evolutionary branch as the HCV 1b subtype sequence, and the nucleotide homology was 96.8%-97.6%; the sequence of one case and the subtype sequence of HCV 3a gene were located in the same evolutionary branch, and the nucleotide homology was 95.2%; the sequence of one case and HCV 6n gene subtype sequence were located in the same evolutionary branch, and the nucleotide homology was 97.9%; One case was located in the same evolutionary branch as the HCV 6u gene subtype sequence, and the nucleotide homology was 98.4%. Conclusions　HCV 1b, HCV 3a, HCV 3b, HCV 6n and HCV 6u genotypes or subtypes of HCV are prevalent in Kunming, and HCV 3b is the most prevalent genotype.

Observation on the Efficacy of Acupuncture plus Intradermal Needle in Treating Mild-to-moderate Post-stroke Depression / 上海针灸杂志

Objective To observe the clinical efficacy of acupuncture plus intradermal needle in treating mild-to-moderate post-stroke depression (PSD). Method Ninty patients with mild-to-moderate PSD were randomized into group A, B and C, with 30 cases in each group. All the groups were intervened by selecting Baihui (GV20), Shenting (GV24) and Yintang (GV29). Group A received acupuncture plus intradermal needle; group B, intradermal needle alone; group C, acupuncture plus sham acupuncture. Hamilton depression rating scale (HDRS) score and Barthel index (BI) score in the three groups were observed before and at the end of the treatment, and the clinical efficacies of the three groups were compared. Result The HDRS score and BI score showed a significant change after the treatment in the three groups (P<0.01). After the treatment, the HDRS score and BI score in the group A and group B was significantly different from those in group C (P<0.05). The total effective rate was 90.0％ in group A versus 86.7％in group B, and 73.3％ in group C; there was a statistically significant difference between group C and group A or B (P<0.01, P<0.05). Conclusion Both acupuncture plus intradermal needle and intradermal needle alone are effective approaches in treating mild-to-moderate PSD. The treatment efficacy of these two methods is similar.

Efficacy of nalbuphine in preventing shivering after neuraxial anesthesia in patients undergoing cesarean section / 中华麻醉学杂志

Objective To evaluate the efficacy of nalbuphine in preventing shivering after neuraxial anesthesia in patients undergoing cesarean section.Methods Eighty American Society of Anesthesiologists physical status Ⅰ or Ⅱ patients,aged 20-35 yr,weighing 55-80 kg,undergoing elective cesarean section under neuraxial anesthesia,were divided into nalbuphine group (group N,n=40) and control group (group C,n=40) using a random number table method.After delivery,nalbuphine 0.1 mg/kg was intravenously injected immediately before clamping the umbilical cord in group N,and the equal volume of normal saline was given instead in group C.Ramsay sedation score was recorded before giving nalbuphine,at 5 min after giving nalbuphine,and at the end of surgery.The development of shivering was recorded from the end of nalbuphine administration until the end of surgery,and the shivering intensity was estimated using Wrench grading.The development of over-sedation,nausea and vomiting,bradycardia,hypotension and dizziness was recorded from the end of nalbuphine administration until the end of surgery.Results Compared with group C,Ramsay sedation scores were significantly increased at 5 min after giving nalbuphine and at the end of surgery,the incidence of shivering was decreased,the shivering intensity was reduced (P＜0.05),and no significant change was found in the incidence of adverse reactions in group N (P＞ 0.05).Conclusion Nalbuphine can prevent the occurrence of shivering after neuraxial anesthesia in patients undergoing cesarean section.

Effect of Ulinastatin Combined with Pantoprazole on Inflammatory Factors and Gastrointestinal Tract in Patients Undergoing Cardiopulmonary Bypass Cardiac Surgery / 医药导报

Objective To investigate the effect of ulinastatin combined pantoprazole on inflammatory factors and gastrointestinal tract in patients undergoing cardiopulmonary bypass ( CBP) cardiac surgery. Methods A total of 200 patients who suffered rheumatic heart disease were scheduled for valve replacement surgery with CPB, were randomly divided into four groups:control group (CON),ulinastatin (UTI),pantoprazole groups (PTZ) and ulinastatin+pantoprazole groups(UTI+PTZ),50 cases in each group.Before CBP,group UTI was given ulinastatin 10 000 U·kg-1,group PTZ was given pantoprazole 40 mg,group UTI+PTZ was given ulinastatin 10 000 U·kg-1and pantoprazole 40 mg,group CON was given 0.9% sodium chloride soution.The gastric mucosa pHi and blood samples would be collected in all four groups at the preoperative (t1),CPB 30 min (t2),after CBP (t3),6 h after surgery (t4),24 h (t5) five time points.The IL-6 and TNF-α would be detected by enzyme linked immunosorbent (ELISA) method,and abdominal distension,abdominal pain,hematemesis,black and defecate occult blood test positive for digestive tract related complications would be collected after the surgery 1,2 days. Results The concentration of TNF-α and IL-6 at t2,t3, t4,t5were higher than those at t1in all four groups(P<0.05).Compared with CON group,the concentration of TNF-α and IL-6 at t2, t3,t4,t5in UTI,PTZ and UTI+PTZ group were significantly decreased (P<0.05).The concentration of TNF-α and IL-6 in UTI and UTI+PTZ group were better than in PTZ group.The pHi at t2,t3,t4was lower than that at t1in four groups(P<0.05),and pHi at t5 was obviously lower than that at t1in group CON (P<0.05).The pHi at t2,t3,t4in UTI,PTZ and UTI+PTZ group was higher than that in CON group ( P<0. 05), and pHi in UTI+PTZ group was better than that in UTI and PTZ group. The postoperative gastrointestinal complications in CON group were higher than those in UTI,PTZ and UTI+PTZ group (P<0.05). Conclusion Ulinastatin combined with pantoprazole for patients undergoing CPB heart surgery,can significantly reduce the release of TNF-α and IL-6、increase gastric pHi and reduce the incidence of gastrointestinal complications.

Changes of autophagy in rat lung tissue after acute spinal cord injury / 中华创伤杂志

Objective To investigate the changes of autophagy in rat lung tissues after acute spinal cord injury (ASCI) and the possible mechanisms.Methods Sixty-three female SD rats were divided into sham operation group (n =21) and spinal cord injury group (n =42),according to the random number table.The modified Allen method with the impact energy of 10 × 25 g · mm at the T10 vertebra was used for preparation of ASCI models.The rats were sacrificed at 6,12,24,48,72 hours,1 and 2 weeks after injury.Lung tissue damage and apoptosis were detected by HE staining and TUNEL method.The changes of autophagy and expressions of LC3-Ⅱ,P62,Beclin-1,interleukin (IL)-17A and Bcl-2 in lung were detected by Western blot and immunofluorescence.Results The pulmonary alveoli maintained normal structure in sham operation group,with no inflammation or pulmonary hemorrhage.Slight lung tissue damages were observed in spinal cord injury group at 12 h postinjury.Alveolar stroma widening,inflammatory infiltration,hemorrhage,and alveolar collapse became ingravescent at 24-72 hours postinjury.Numbers of apoptotic cells in spinal cord injury group were 551.22 ± 135.94,905.11 ±92.64,and 141.78 ± 30.86 respectively at 24,72 hours and 1 week postinjury,and were significantly increased at 24 and 72 hours postinjury,compared with sham operation group (P ＜ 0.05).Expression of LC3-II in spinal cord injury group was increased at 24-72 hours postinjury,compared with sham operation group (P ＜ 0.05).Expression of P62 in spinal cord injury group was up regulated at 24-72 hours postinjury,compared with sham operation group (P ＜ 0.05).Expression of Beclin-1 in spinal cord injury group was increased at 24 h postinjury and then dropped at 48-72 hours,compared with sham operation group (P ＜ 0.05).Expression of IL-17A in spinal cord injury group was increased at 24-48 hours,compared with sham operation group (P ＜ 0.05).Expression of Bcl-2 in spinal cord injury group was increased from 24 hours to 72 hours,compared with sham operation group (P ＜ 0.05).Conclusion Autophagosome formation is increased and accumulated in the lung tissues after ASCI,which might be related to the increased interaction between Beclin-1 and Bcl-2 because of the up regulation of Bcl-2 by IL 17A,ultimately leading to the inhibition of autophagy.

Tumor necrosis factor-α and transforming growth factor-β balance liver stem cell differentiation in cholestatic cirrhosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the changes of tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) in mice with cholestatic cirrhosis and their role in regulating the balance of liver stem cell differentiation.</p><p><b>METHODS</b>Balb/c mice were subjected to bile duct ligation (BDL), and serum biochemical parameters were measured and hepatic histopathology was observed using HE staining to evaluate the modeling of cholestatic cirrhosis. Immunohistochemistry and Western blotting were used to detect the changes of TNF-α and TGF-β in the mice after modeling. Mouse embryonic hepatic stem cells (HP14-19) were treated with different concentrations of TNF-α and TGF-β, and the cell differentiation was assessed using Western blotting, real-time PCR, and PAS staining.</p><p><b>RESULTS</b>The mice receiving BDL showed significantly increased blood biochemical parameters (P<0.05), and HE staining revealed obviously increased collagen fibers in the liver with significantly increased expressions of TNF-α and TGF-β (P<0.05). In HP14-19 cells, induction with TNF-α and TGF-β for 3 days did not cause significant changes in cell differentiation, but induction for 5 days resulted in significantly increases intensity of PAS staining in the cells. The cells induced with 20, 40, and 80 ng/mL TNF-α for 5 days exhibited a significantly stronger expression of cytokeratin 18 than cytokeratin 19 (P<0.05), while induction with 20, 40, and 80 ng/mL TGF-β produced opposite changes in cytokeratin 18 and cytokeratin 19 expressions. Further induction of the cells with TNF-α and TGF-β for 10 days, did not alter the expression patterns of cytokeratin 18 and cytokeratin 19 observed on day 5, but their protein expression levels and PAS staining intensity of the cells were enhanced and their mRNA expressions became lowered.</p><p><b>CONCLUSION</b>Common bile duct ligation can induce conditions simulating cholestatic cirrhosis in mice. TNF-α and TGF-β are elevated in cholestatic cirrhosis and play opposite roles in regulating the differentiation balance of liver stem cells: the former promotes the differentiation of liver stem cells into hepatocytes, while the latter promotes the cell differentiation into colangiocytes.</p>

Changes in autophagy during maturation and differentiation of Hepa1-6 cells induced by all-trans retinoic acid / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of different concentrations of all-trans retinoic acid (ATRA) on the maturation, differentiation and autophagy of Hepa1-6 cells.</p><p><b>MONTHOD</b>Hepa1-6 cells were treated with 0.1, 1, and 10 µmol/L ATRA, and the changes in the expressions of hepatic specific markers were detected using real-time PCR and Western blotting. Indocyanine green (ICG) and periodic acid-schiff (PAS) staining was used to assess the functional maturation of Hepa1-6 cells, and the cell-cell junction and autophagy were observed under transmission electron microscopy to determine the optimal concentration of ATRA for treatment. The expressions of autophagy-related markers in the cells were detected using Western blotting, and confocal microscopy was used to observe the autophagic flow in the cells transfected with ptfLC3 plasmid.</p><p><b>RESULTS</b>Compared with the control cells, the hepatocytes treated with ATRA showed a concentration-dependent decrease in AFP expression and increase in the expressions of ALB, CK18, TAT and ApoB. ICG and PAS staining revealed significantly increased number of positive cells after ATRA treatment. Following ATRA treatment, the cells exhibited obviously increased tight junctions, cytoskeleton and number of autophagosomes under transmission electron microscopy. ATRA treatment resulted in significantly increased the expressions of autophagy-related markers LC3-II, Beclin-1, RAB7 and P62 and also an increased ratio of LC3-II/LC3-I(P<0.05). Confocal microscopy revealed obviously increased green and red spots in the cells after ATRA treatment.</p><p><b>CONCLUSION</b>ATRA can induce the maturation and differentiation and enhance the level of autophagy in Hepa1-6 cells.</p>

Twist regulates proliferation, migration and invasion of osteosarcoma cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of Twist in regulating the proliferation, migration, and invasion of osteosarcoma cells with different levels of malignancy.</p><p><b>METHODS</b>The baseline expressions of Twist in 3 different osteosarcoma cell lines (143B, MG63 and TE85) were detected using real-time PCR and Western blotting. The cells were infected with the recombinant adenoviruses Ad-Twist or Ad-siTwist for Twist overexpression or knockdown, respectively, and the cell growth curves were drawn to assess the cell proliferation. The migration abilities and invasiveness of the cells were evaluated using wound healing assay and Transwell assay. Luc-labeled 143B cells infected with Ad-Twist or Ad-siTwist were intrathecally injected to establish nude mouse models bearing osteosarcoma xenografts, in which the tumor formation was monitored using living body imaging technique.</p><p><b>RESULTS</b>The baseline expressions of Twist in the 3 osteosarcoma cells were significantly higher than that in C3H10 cells (P<0.05). Twist expression was the highest in 143B cells followed by MG63 cells, and was the lowest in TE85 cells, indicating its positive correlation with the level of malignancy of the osteosarcoma cells. Ad-Twist or Ad-siTwist infection efficiently enhanced or lowered Twist expressions at both mRNA and protein levels in osteosarcoma cells (P<0.05). Twist overexpression resulted in enhanced proliferation, migration and invasion abilities of osteosarcoma cells, and Twist knockdown obviously inhibited the cell proliferation, migration and invasion. In nude mice, 143B cells with Twist overexpression showed accelerated tumor formation compared with the control cells, while Twist knockdown significantly inhibited the tumor formation ability of the cells.</p><p><b>CONCLUSION</b>Twist overexpression can promote the proliferation, migration, invasion and tumorigenicity of osteosarcoma cells.</p>

Acetylated Histone Expressions of the Primary Hippocampal Neurons in Rats Reduced by siCBP Lentivirus / 生物医学工程学杂志

This study aims to construct the recombinant lentivirus vector containing specific small interfering RNA (siRNA) targeting rat CREB binding protein(CBP)gene and to identify its function of inhibiting the expressions of acetylated histone in primarily cultured hippocampal neurons. Firstly, we constructed four kinds of recombinant lentivirus siCBP. And then we used them to infect the primarily cultured hippocampal neurons, and performed real-time PCR, western blot respectively to detect the expressions of CBP. Afterwards, the most effective lentivirus siCBP was used to infect the primarily cultured hippocampal neurons, and then the HAT activity and protein expressions of acetylated histone Ac-H3, Ac-H4 of the neurons were examined. By using PCR, endonuclease cutting and gene sequencing, we confirmed that the target genes were correctly cloned in lentivirus vector. Besides, CBP mRNA and protein expressions in neurons were found to be with varying degrees of decreases after infections of the four kinds of lentivirus siCBP. Furthermore, the representative and most effective lentivirus GR806 could effectively inhibit the HAT activity and the protein expressions of Ac-H3, Ac-H4 in neurons. It provides the experimental basis for the subsequent application of siCBP to clarify the effects and corresponding molecular mechanism of the CBP-dependent histone acetylation on learning and memory function in hippocampus.

Effects of Plasmid Fibroblast Growth Factor-2 Magnetic Chitosan Gelatin Microspheres on Proliferation and Differentiation of Mesenchymal Stem Cells / 生物医学工程学杂志

The purpose of this study is to investigate the effect of superparamagnetic chitosan FGF-2 gelatin microspheres (SPCFGM) on the proliferation and differentiation of mouse mesenchymal stem cells. The superparamagnetic iron oxide chitosan nanoparticles (SPIOCNs) were synthesized by means of chemical co-precipitation, combined with FGF-2. Then The SPCFGM and superparamagnetic chitosan gelatin microspheres (SPCGM) were prepared by means of crosslinking-emulsion. The properties of SPCFGM and SPIONs were measured by laser diffraction particle size analyser and transmisson electron microscopy. The SPCFGM were measured for drug loading capacity, encapsulation efficiency and release pharmaceutical properties in vitro. The C3H10 cells were grouped according to the different ingredients being added to the culture medium: SPCFGM group, SPCGM group and DMEM as control group. Cell apoptosis was analyzed by DAPI staining. The protein expression level of FGF-2 was determined by Western blot. The proliferation activity and cell cycle phase of C3H10 were examined by CCK8 and flow cytometry. The results demonstrated that both of the SPIOCNs and SPCFGM were exhibited structure of spherical crystallization with a diameter of (25 ± 9) nm and (140 ± 12) μm, respectively. There were no apoptosis cells in the three group cells. Both the protein expression level of FGF-2 and cell proliferation activity increased significantly in the SPCFGM group cells (P < 0.05). The SPCFGM is successfully constructed and it can controlled-release FGF-2, remained the biological activity of FGF-2, which can promote proliferation activity of C3H10 cells, and are non-toxic to the cell.

A family study of mandibuloacral dysplasia with type A lipodystrophy / 临床儿科杂志

Objective To study the gene mutations and clinical features of mandibuloacral dysplasia with type A lipodystrophy (MADA) in a Chinese family. Methods The information of 5 family members including 2 siblings suspected atyp-ical progeria was assembled. Genomic DNA was extracted from peripheral blood of 5 family members, the 12 exons of LMNA gene were ampliifed by PCR and then the PCR products were directly sequenced and analyzed by using Blast software online. The SIFT and PolyPhen-2 software were used to predict the harmfulness of mutations. Results The 2 siblings were clinically diagnosed as MADA. Heterozygous c.1579C>T (p.Arg527Cys) and c.1583C>T (p.Thr528Met) mutations were detected in this family. The father carried c.1583C>T (p.Thr528Met) mutation, the mother carried c.1579C>T (p.Arg527Cys) mutation, and their normal daughter were all heterozygous carriers with c.1583C>T (p.Thr528Met) mutation. Compound heterozygous c.1579C>T (p.Arg527Cys) and c.1583C>T (p.Thr528Met) mutations in 2 siblings led to MADA. The MADA showed an autosomal re-cessive inheritance pattern in this family. Conclusions The 2 siblings with MADA in this family were caused by compound heterozygous mutations in LMNA gene.

Effect of BMP-2 and -3 overexpression on osteogenic and chondrogenic differentiation of prenatal mouse intervertebral disc cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein (BMP)-2 and -3 over expressions on chondrogenesis and osteogenesis of prenatal mouse intervertebral disc cells and provide experimental evidences for the application of BMPs in the therapy of disc diseases.</p><p><b>METHODS</b>The prenatal mouse intervertebral disc cells were infected with a recombinant adenovirus expressing BMP-2 and BMP-3 for 5-7 days, and the expressions of collagen type I (Col I), collagen type II (Col II), aggrecan, osteocalcin, osteoprotegerin and osteopontin mRNAs were detected with RT-PCR. The expression of cartilage matrix was evaluated with toluidine blue staining, and alkaline phosphatase (ALP) activity was detected with ALP reading and ALP staining.</p><p><b>RESULTS</b>BMP-2 and -3 overexpression did not enhance chondrogenesis and osteogenesis of annulus fibrosus (AF) cells or cause significant increases in the expressions of Col I, Col II or aggrecan mRNA in nucleus pulposus (NP) cells. Adenovirus-mediated overexpression of BMP-2 and BMP-3, however, promoted osteogenesis of NP cells and significantly increased the expression of osteocalcin mRNA; the overexpression of BMP-2, but not BMP-3, enhanced the mRNA expressions of osteoprotegerin and osteopontin. Toluidine blue staining demonstrated that BMP-2 and BMP-3 overexpression did not obviously affect the secretion of cartilage matrix. In NP cells, BMP-2 and -3 overexpression significantly enhanced ALP activity, which was not observed in AF cells.</p><p><b>CONCLUSION</b>Adenovirus-mediated BMP-2 and -3 overexpression can promote the osteogenic differentiation of NP cells but can not affect osteogenesis of AF cells or chondrogenesis of NP cells.</p>

Construction of a luciferase reporter vector containing response element of activator protein 2α and its application in study of osteogenetic differentiation / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a luciferase reporter vector containing the response element of transcription protein AP2α for screening the effect of bone morphogenetic proteins (BMPs) on the transcriptional activity of AP2α.</p><p><b>METHODS</b>Four tandem-linked response elements of AP2α were cloned to the pBGLuc luciferase reporter gene plasmid, which was digested with Bam HI and Mlu I to construct pBGLuc-AP2α-RE vector. The recombinant adenovirus Ad-AP2α and its dominant negative mutant Ad-dnAP2α were used to infect mouse mesenchymal stem cells C3H10; the changes in cellular AP2α mRNA and protein expressions were detected by real-time PCR and Western blotting, and electrophoretic mobility shift assay (EMSA) was carried out to assess the DNA-binding ability of AP2α. C3H10 cells were transfected with pBGLuc-AP2α-RE vector, and AP2α transcriptional activity was measured using luciferase reporter gene assay. In pBGLuc-AP2α-RE vector-transfected C3H10 cells infected with Ad-BMPs, luciferase reporter gene assay was performed to screen the effect of BMPs on AP2α transcriptional activity.</p><p><b>RESULTS</b>The results of PCR, enzyme digestion and sequencing all confirmed correct cloning of AP2α-RE into pBGLuc-AP2α-RE luciferase reporter vector, and Ad-AP2α infection significantly increased AP2α expression and its DNA binding ability. The dominant negative mutants expressed the corresponding mutants, and EMSA results showed that Ad-dnAP2α-δbHLH significantly lowered while Ad-dnAP2α-δTAD enhanced the DNA-binding ability of AP2α. AP2α over-expression promoted AP2α transcriptional activity, which was suppressed by the two dominant negative mutants. AP2α transcriptional activity increased in the cells infected with the recombinant adenovirus BMPs, especially in cells with BMP9 infection.</p><p><b>CONCLUSIONS</b>The luciferase reporter vector containing the response element of AP2α we constructed allows detection of AP2α transcriptional activity. BMP9 can significantly enhance AP2α transcriptional activity.</p>

Recombinant adenovirus expressing siRNA is generated to inhibit the expression of RARbeta in rat mesenchymal stem cells treated by all-trans retinoic acid / 生物工程学报

To construct the recombinant adenovirus vector expressing specific siRNA for rat retinoic acid receptor-beta (RARbeta) gene, and to detect its effect on RARbeta expression and neuronal differentiation of all-trans retinoic acid (ATRA) treated mesenchymal stem cells (MSCs). First, we designed four pairs of siRNA sequence for rat RARbeta gene and annealed complementary oligonucleotides in vitro, then cloned double-stranded DNA in pSES-HUS vector containing U6/H1 double-promoter and recombinated with the backbone vector to construct pAd-siRARbeta plasmid. We infected MSCs by using adenovirus Ad-siRARbeta which was packaged in HEK293 cell line, then performed Real-time, Western blotting and immunoflourencence to detect the expression of RARbeta. We used combination of ATRA and MNM to induce MSCs into neural-like cells, then performed Real-time PCR and immunoflourencence to detect neuronal specific markers of induced neural cells. By using PCR, endonuclease cutting and gene sequencing, we confirmed that the target genes were correctly cloned in adenovirus vector. We could observe more than 60% RFP-positive MSCs at 24 h after adenovirus infection. The expression of RARbeta was significantly increased to 16.5 +/- 2.34 fold in ATRA treated MSCs (P < 0.05) and located in nucleus. Three of four pairs siRNA could effectively inhibit the expression of RARbeta with inhibition efficacy of (66.26 +/- 9.12)%, (48.70 +/- 5.78)%, (64.09 +/- 0.53)% (P < 0.05), especially siRNA-pool group with inhibition efficacy of (78.09 +/- 4.24)% (P < 0.01). Combination of ATRA and MNM induced MSCs into neural-like cells which expressed neuronal specific markers, Nestin, NSE, MAP-2, and Tau. Immunoflourencence result showed that about 50-88 present of cells were positive for Nestin, NSE, Tjul, however, adenovirus medicated expression of siRARbeta could effectively inhibit the expression level of neural specific proteins and the ratio of positive stained cells (P < 0.05). Therefore, we successfully constructed the recombinant adenovirus vector containing siRNA for rat RARP gene, adenovirus could effectively infect MSCs and inhibit the expression of induced RARbeta in ATRA treated MSCs, then inhibit neuronal differentiation of MSCs.

Screening and identification of effective small interfering RNAs against bone morphogenetic protein-9 / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen specific small interfering RNA (siRNA) targeting mouse BMP9 gene and identify its function in BNLCL.2 fetal liver cells and C3H10 cells.</p><p><b>METHODS</b>Four pairs of double-stranded DNA fragments for silencing mouse BMP9 were annealed in vitro and cloned into pSOS-BMP9 vector with BMP9 gene to construct pSOS-simBMP9 plasmid. The 4 pSOS-simBMP9 plasmids were separately transfected in HEK293 cells via Lipofectamine, and the gene silencing efficiency was assessed by GFP detection. BNLCL.2 fetal liver cells were infected with the constructed adenovirus simBMP9s, and their BMP9 expression was detected with RT-PCR and Western blotting. C3H10 cells were co-infected with Ad-simBMP9 and Ad-BMP9, and the inhibitory effect on BMP9-induced osteoblasts was evaluated by alkaline phosphatase (ALP) activity.</p><p><b>RESULTS</b>GFP expression in the two simBMP9 groups was significantly decreased in HEK293 cells, and the endogenous expression of BMP9 was reduced by 50%-70% by adenovirus-mediated simBMP9 in the fetal liver cells. ALP activity in C3H10 cells was significantly higher in BMP9 group than in the control group (P<0.01), while the activity of the two Ad-simBMP9-infected groups was significantly lower than that in Ad-BMP9-infected group (P<0.01).</p><p><b>CONCLUSION</b>Two specific siRNA targeting mouse BMP9 gene have been obtained, which can effectively inhibit both endogenous and exogenous expressions of BMP9 to facilitate the study of the mechanisms of BMP9 in liver cell differentiation.</p>

Sensitivity of HepG2 cells with stably expressing HBx via rosiglitazone and its mechanism / 肿瘤

Objective: To establish a HepG2 cell line with stable expression of x protein of hepatitis B virus (HBx), detect its sensitivity to rosiglitazone, and explore the mechanism for the role of the interaction of HBx with peroxisome proliferato-activated receptor γ(PPARγ) in hepatocarcinogenesis. Methods: This work constructed pIRES2-HBx eukaryotic expression plasmid, screened HepG2 cell clone with stable expression of HBx. The sensitivity of HBx-expressing HepG2 cells to rosiglitazone was assessed by MTT assay. The change in PPARγ location was detected by immunocytochemistry. The mRNA and protein expressions of PPARγ were determined by RT-PCR and Western blotting respectively. Results: This work successfully constructed pIRES2-HBx eukaryotic expression plasmid and obtained the HepG2 cell line with stable expression of HBx. The inhibitory effect of rosiglitazone on HBx-expressing HepG2 cells decreased obviously, but the inhibitory effect was partly reversed when the cells were pretreated with PD98059, an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1). The difference in PPARγ expression was not significant. But the location of PPARγ was changed. It moved from nucleus to cytoplasm. Conclusion: HBx reduces the sensitivity of HepG2 cells to rosiglitazone. The possible mechanism is that HBx changes the site-specific location of PPARγ and decreases its binding ability to DNA, thus influence the binding of PPARγ to its ligand and the activity of PPARγ through phosphorylation pathway.

Construction and identification of recombinant adenovirus vector encoding ?-galactosidase with modified AdEasy system / 第三军医大学学报

Objective To construct a recombinant adenovirus encoding ?-galactosidase gene(?-gal),which may be used to treat the lactose intolerance caused by lactase deficiency,with modified AdEasy system.Methods The sequence fragment of ?-gal was cloned into the shuttle plasmid pAdTrack-CMV,and the homo-logous recombination was completated with backbone plasmid pAdEasy1 in the E.coli BJ5183 to construct the recombinant adenoviral plasmid.The adenoviruses were packaged and amplified in the HEK293 cells mediated by liposome.The viral titre was measured by limiting dilution assay and the transfection efficiency was observed by X-gal histochemical staining.Results The recombinant adenovirus with ?-gal was constructed successfully and the viral titre was(2.5~4.0)?1011EFU/ml.More than 70% HEK293 cells were transfected when the MOI was 10.Moreover,more than 65% cells were blue after X-gal histochemical staining.Conclusion Recombinant adenovirus carrying ?-gal with high titre and efficient transfection is constructed successfully by AdEasy system,which supplies valuable experimental basis for the gene therapy to the lactose intolerance caused by lactase deficiency.

Construction and identification of recombinant adenovirus vector containing human ?-catenin / 重庆医科大学学报

Objective:To construct the recombinant adenovirus vector containing human?-catenin gene by homogenous recombination in E.coli for later research of the mechanism of Wnt/Wingless signal pathway in tumorigenesis.Methods:The target gene was amplified from pcmv-?NS-FC plasmid by PCR,then cutted by two endonucleases and directional cloned to the pAd track-cmv vector.Afterward,the correct recombinant was linearized by PmeI,following co-transformation with the backbone vector pAd Easy-1 in E.coli BJ5183.The homologous recombinant was linearized by PacI,and then transfected into HEK293 cell line to pack the adenovirus.Results:Through PCR,endonuclease cutting and gene sequencing,the target gene was verified to be correctly cloned in adenovirus vector.The high expression of green fluorescence protein in 293 cell line was found under fluorescent microscope.Conclusion:The recombinant adenovirus vector containing human ?-catenin gene was successfully constructed and the adenovirus was packaged in HEK293 cell line.