The effects and mechanisms of trimethylamine-N-oxide on insulin sensitivity in insulin target cells / 药学学报

Gut microbial metabolite trimethylamine-N-oxide (TMAO) is associated with type 2 diabetes (T2DM). Decreased insulin sensitivity is a significant etiological factor of T2DM. Adipocytes, myocytes, and hepatocytes are the three major target cells for insulin. This study aims to investigate the effects and mechanisms of TMAO on the insulin sensitivity of these target cells. Research results indicate that in different ages of db/db diabetic mice, plasma TMAO levels were increased. TMAO significantly inhibits the insulin signaling pathways in these three major insulin target cells, reduces glucose uptake in 3T3-L1 adipocytes and L6 myocytes and downregulates genes related to gluconeogenesis in primary mouse hepatocytes. Furthermore, in mice with normal insulin sensitivity, elevating plasma TMAO levels to those seen in db/db mice using a minipump results in impaired glucose tolerance and hyperinsulinemia. All animal experiments were carried out with approval of the Experimental Animal Welfare Ethics Committee of the Institute of Materia Medica (Chinese Academy of Medical Sciences and Peking Union Medical College). Mechanistic studies suggest that TMAO exposure increases the levels of endoplasmic reticulum stress-related proteins in these three major insulin target cells. In summary, TMAO directly attenuates insulin sensitivity in insulin target cells, and its mechanism of action may involve enhancing endoplasmic reticulum stress.

Analysis of the of Pb, Cd and As in decoction of Lindera aggregata (Sims) Kosterm. by PBET digestion in vitro/Caco-2 cell model and their cumulative risk assessment / 药学学报

Inductively coupled plasma mass spectrometry (ICP-MS) was applied to determine the concentrations of lead (Pb), cadmium (Cd) and arsenic (As) in Lindera aggregata (Sims) Kosterm. The physiologically based extraction test (PBET) digestion in vitro/Caco-2 cell model was established to investigate the bioaccessible contents of Pb, Cd and As in decoction of Lindera aggregata (Sims) Kosterm. The target-organ toxicity dose modification of HI method (TTD) was used to evaluate the cumulative risk caused by the combined exposure of the total levels of Pb, Cd and As in Lindera aggregata (Sims) Kosterm. and the bioaccessible contents in the decoction. The results showed that the total contents of Pb, Cd and As in 4 batches of samples were in the range of 2.901-3.872, 1.299-1.800 and 0.062-0.216 mg·kg-1, respectively. After transportation by Cacco-2 cells, the bioaccessible contents of Pb, Cd, and As in the decoction were in the range of 0.045-0.080, 0.070-0.112 and 0.004-0.018 mg·kg-1. The results of risk assessment showed that calculated by the total amounts of heavy metals in the Lindera aggregata (Sims) Kosterm., for the end points of nervous system, the cumulative risks of co-exposure of heavy metals in 3 batches of samples were of concern. After decoction and transportation by Caco-2 cells, for the end points of cardiovascular system, blood, nervous system, kidney and testis, the TTD modification of HI values of all batches of samples were less than 1, and the health risks were acceptable. The study provided methodology basis for a more objective assessment of the health risks of heavy metals and harmful elements in traditional Chinese medicine and for a more scientific limit standard of heavy metals and harmful elements.

The effect of ventriculoscopy combined with laparoscopy in ventriculoperironeal shunt on the treatment effect, inflammatory factors and immune function in patients with hydrocephalus / 中国医师进修杂志

Objective:To investigate the therapeutic effect of ventriculoscopy combined with laparoscopy in ventriculoperironeal shunt (VPS) on the treatment effect, inflammatory factors and immune function in patients with hydrocephalus.Methods:A retrospective study was conducted on 76 patients with hydrocephalus in Traditional Chinese Medicine Hospital of Jining from March 2017 to September 2019, and they were divided into observation group (39 cases) and control group (37 cases) according to different treatment procedures. The control group performed traditional VPS, and the observation group performed ventricoscopy combined with laparoscopy in VPS. The incidence of postoperative complications and the treatment effect at 6 months after the operation, serum inflammatory factors tumor necrosis factor-α (TNF-α), interleukin (IL)-10, IL-6, immune function indexes CD 3+, CD 4+, CD 4+/CD 8+before and 1 d and 3 d after the operation, Glasgow Outcome Scale (GOS) and Modified Rankin Scale (MRS), National Institutes of Health Stroke Scale (NIHSS), Barthel Index (BI) and Generic Quality of Life Inventory 74 (GQOL-74) before operation, 3 and 6 months after operation were compared between the two groups. Results:The total effective rate in the observation group at 6 months after operation was higher than that in the control group: 94.9% (37/39) vs. 73.0% (27/37), and the difference was statistically significant ( χ2 = 0.848, P<0.05). The GOS scores in the observation group were higher than those in the control group at 3 months and 6 months after surgery: (3.68 ± 0.49) scores vs. (3.02 ± 0.51) scores, (4.17 ± 0.24) scores vs. (3.59 ± 0.43) scores; and the MRS and NIHSS scores were lower than those in the control group: (2.05 ± 0.15) scores vs. (2.84 ± 0.17) scores and (1.62 ± 0.11) scores vs. (2.17 ± 0.14) scores, (12.97 ± 3.82) scores vs. (16.05 ± 4.61) scores and (10.90 ± 2.75) scores vs. (13.84 ± 3.29) scores; and the differences were statistically significant ( P<0.05). The serum levels of TNF-α and IL-6 in the observation group were lower than those in the control group at 1 and 3 d after surgery, and IL-10 level was higher than that in the control group, and the differences were statistically significant ( P<0.05). The CD 3+, CD 4+, and CD 4+/CD 8+ levels in the observation group were higher than those in the control group at 1 and 3 d after operation, and the differences were statistically significant ( P<0.05). The postoperative complications in the observation group was lower than that in the control group: 29.7% (11/37) vs. 10.3% (4/39), and the difference was statistically significant ( χ2 = 4.383, P<0.05). The BI and GQOL-74 scores in the observation group at 3 and 6 months after operation were higher than those in the control group ( P<0.05). Conclusions:The combination of ventricoscopy and laparoscopic in VPS can reduce the impact on immune function by reducing the inflammatory stress response, reduce the occurrence of postoperative complications, and further promote the improvement of prognosis.

Hepatic injury following umbilical vein catheterization in neonates: a report of seven cases and literature review / 中国小儿急救医学

Objective:To study the clinical characteristics of hepatic injury following umbilical vein catheterization, so as to improve the safety and use of umbilical vein catheterization in the newborn.Methods:Data of infants undergoing umbilical vein catheterization during hospitalization in the neonatology department of the Third Affiliated Hospital of Guangzhou Medical University from Jan 1st, 2015 to Dec 31st, 2019 were collected retrospectively.The clinical characteristics, diagnoses and treatments of the infants with hepatic injury following umbilical vein catheterization were summarized and analyzed.And the relative literature was reviewed.Results:A total of 1 721 infants underwent umbilical vein catheterization, and seven cases(0.4%)had hepatic injury, including six cases of exudative necrosis in the liver and one case of hepatic hematoma.There was no significant difference in gestational age[(31.12±2.64)weeks vs.(30.62±2.75)weeks]or birth weight[(1 493±525)g vs.(1 374±473)g]between hepatic injury group and non-hepatic injury group( P>0.05). Clinically, it was found that the tip position of umbilical vein catheter was below the diaphragm(between the 9th and 11th thoracic vertebrae)in all seven cases.Except one case with no obvious clinical symptoms, the other six cases had clinical symptoms, mainly manifested as abdominal distension, repeated gastric retention, decreased bowel sounds, and additional with shock and anemia in hepatic hematoma.Blood sample tests showed elevated C-reactive protein, white blood cell count and transaminase.X-ray examination revealed cystic translucent shadow in the liver, and ultrasound and/or computed tomography examination showed the lesion in more detail.After removal of the catheter and administration of antibiotic, the hepatic injury recovered gradually. Conclusion:The hepatic injury following umbilical vein catheterization in neonates is not common.The catheter tip under the diaphragm is the main risk factor.And the diagnosis is dependent on imaging examination.

Effect of weight growth velocity in the early stage after birth on neurodevelopment of preterm infants at the corrected age of 12 months / 中国当代儿科杂志

OBJECTIVE@#To study the effect of weight growth velocity in the early stage after birth on the neurodevelopment of preterm infants at the corrected age of 12 months.@*METHODS@#Related data were collected from the preterm infants who were discharged from the Department of Neonatology, Third Affiliated Hospital of Guangzhou Medical University, from July 1, 2015 to December 31, 2019 and were followed up at the outpatient service of high-risk infants. According to the weight growth velocity from birth to the corrected gestational age of 40 weeks, the infants were divided into two groups: low velocity [< 10 g/(kg·d); @*RESULTS@#At the corrected age of 12 months, the low velocity group had a significantly lower score of fine motor (@*CONCLUSIONS@#The weight growth velocity from birth to the corrected age of 40 weeks affects the development of fine motor and language in preterm infants at the corrected age of 12 months; however it needs to be further verified by large-sample studies.

Content Determination of Lupanone and Stigmasterol in Different Parts of Musa basjoo by UPLC / 中国药房

OBJECTIVE：To esta blish a method for determining the contents of lupenone and stigmasterol in the rhizome ，stem and leaf of Mosa basjoo from the same plant ，and to provide reference for the substitute resource for the effective components of M. basjoo . METHODS ：UPLC method was adopted. The determination was performed on Zorbax Rrhd Eclipse Plus C 18 column （100 mm×2.1 mm，1.8 μm）with mobile phase consisted of acetonitrile-methanol （78.5∶21.5，V/V）. The detection wavelength was set at 210 nm；the flow rate was 0.15 mL/min；the column temperature was 30 ℃ and the sample size was 1 μL. The results of content determination of lupinone and stigmasterol in the rhizome ，stem and leaf of 9 batches of M. basjoo from the same plant were analyzed by the methods of comparative analysis between groups ，principal component analysis and cluster analysis. RESULTS：The mass concentration of lupenone and stigmasterol had a good linear relationship with the corresponding peak area within the range of 11.16-357.10 and 8.83-160.40 g/mL（R2 were 0.999 2 and 0.999 1，respectively）. RSDs of precision ， repeatability and stability tests were all less than 3％. The average recovery rates of lupenone and stigmasterol were 101.44％ and 98.32％，and the RSDs were 1.77％ and 1.81％（n＝6），respectively. The average contents of lupenone and stigmasterol in stems of M. Basjoo were significantly higher than those of rhizome and leaves of M. basjoo （P＜0.05）. There was no statistical significance in the contents of lupenone and stigmasterol between stem and leaf of M. basjoo from same plant （P＞0.05）. Results of principal component analysis showed that the contents of lupanone and stigmasterol were different in rhizome ，stem and leaf of M. basjoo from the same plant. Rhizome ，stem and leaf of M. basjoo were divided into three types through cluster analysis ，among which the rhizome had significant difference with the other two parts. CONCLUSIONS ：The method is simple ，rapid，specific， reproducible and accurate. It can be used for the content determination of lupenone and stigmasterol in different parts of M. basjoo . The stem of M. basjoo can replace the rhizome of M. basjoo as the source of lupinone and stigmasterol.

Correlation between Serum Levels of Hcy,Cys C and Cognitive Impairmentin Patients with Chronic Kidney Disease / 现代检验医学杂志

Objective To investigate the correlation between serum levels of homocystein (Hcy),Cystatin C (Cys C)and cognitive impairment in patients with chronic kidney disease (CKD).Methods 78 patients with CKD from January 2016 to May 2017 in Hanzhong People's Hospital in Shaanxi were enrolled for the study and divided into stage 3 groups (n 27),stage 4 group (n=28) and stage 5 group (n =23) according to stage of CKD.Montreal cognitive assessment scale (MoCA) was performed to evaluate the cognitive level of patients,and they were divided into cognitive impairment group and normal group.Serum levels of Hcy,Cys C,creatinine (SCr) and urea nitrogen (BUN) were compared among groups,and the correlation between them and cognitive level was analyzed.Results ①Serum levels of Hcy,Cys C,SCr and BUN in stage 3 group were significantly lower than that in stage 4 and 5 group,and these indexes in stage 4 group were significantly lower than that in stage 5 group (F=19.221～66.539,all P＜0.05).The score of MoCA in stage 3 group was significantly lower than that in stage 5 group,and the incidence of cognitive impairment was significantly higher than that in stage 5 group (F/x2 =3.993～6.358,all P＜0.05).②The serum levels of Hcy,Cys C,SCr and BUN in cognitive impairment group were significantly higher than in normal group (t =2.198 ～ 3.835,all P＜ 0.05).③ Pearson analysis result showed that MoCA score was negatively correlated with serum Hcy,Cys C and SCr (r=-0.370,-0.392,-0.253,P＜0.05),but there was no sig nificant correlation between score and BUN (r=-0.211,P＞0.05).④For prediction of cognitive impairment,area under the receiver operating curve of serum levels of Hcy,Cys C,SCr and BUN were 0.818,0853,0.751 and 0.709 (P＜0.05).Conclusion Serum levels of Hcy,Cys C were significantly negatively correlated with cognitive function in patients with CKD,whose predictive value were higher than that ot traditional renal function indexes such as SCr and BUN.

Effect of Cholecalciterol Cholesterol Emulsion on Liver of Zymosan-induced Multiple Organ Failure in Mice / 中国医科大学学报

Objective To study the effect of cholecalciterol cholesterol emulsion(CCE)in the zymosan(Z)-induced acute hepatic injury. Meth-ods A total of 36 C57 BL/6 mice were randomly divided into 4 groups,namely negative control(CON)group,CCE group,Z group and CCE+Z group,respectively. There were 9 mice in each group. Mice from CON group and Z group were fed with pure water. Mice from CCE group and CCE+Z group were fed with cholecalciterol cholesterol emulsion 20μL dissolved in 200 mL pure water which was kept in darkness. After 14 days, Z group and CCE+Z group were injected with zymosan at a dose of 500 mg/kg. After 18 hours,all the mice in each group were sacrificed. The liver tissues were harvested for histopathological examination. The serum ALT levels were determined. The molecular expression of IL-6 and IL-18 in liv-er tissue of mice were evaluated by Western blotting and real-time quantitative PCR method. Results The results of histopathological examination showed that liver tissue damage in CCE+Z group was lighter than that of Z group ,and heavier than that of the CON group. Compared to the CON group,Z group had the highest serum ALT level,followed by CCE+Z group,while in Z group was significantly lower than that in CON group(all P<0.05). The expression of IL-6 and IL-18 protein and mRNA showed level of Z group was apparently higher than those of CON group and CCE+Z group(all P<0.05). Conclusion Cholecalciterol cholesterol emulsion can play certain protective effect on zymosan-induced liver injury in mice.

The relationship between vitamin A and pulmonary surfactant protein with neonatal pulmonary function / 中国小儿急救医学

Objective To investigate the correlation between vitamin A and surfactant protein (SP)-B, SP-C in human body,and to explore the effects on lung development and pulmonary function of neonates. Methods We collected the blood samples of 170 pregnant women and umbilical cord serum of their neonatal babies. The levels of vitamin A in pregnant women and their neonatal babies,and the levels of SP-B and SP-C in neonatal umbilical cord serum were detected by ELISA. We conducted a follow-up by standard telephone questionnaire,which we concerned was the number of respiratory tract infection within six months,in order to assess the neonatal pulmonary functions. Results (1) There was a positive correlation between the vitamin A levels in neonatal umbilical cord blood and in the blood of pregnant women(r=0. 866,P<0. 05). (2) There was a positive correlation between the vitamin A levels in neonatal umbilical cord blood and the levels of SP-B,SP-C in the blood(r=0. 817,P<0. 05). (3)In the follow-up of 170 cases of infants within six months,three cases with pneumonia hospitalized more than once,but no respiratory distress syndrome hap-pened. Conclusion Vitamin A can be used as an important biological marker to evaluate the neonatal pul-monary maturity. If we detect the vitamin A levels of pregnant women,increase the intake of vitamin A,we can improve the content of SP-B,SP-C,improve the development of neonatal lung function in growth.

TXNDC5 mediates serum starvation-induced proliferation inhibition of HeLa cell / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the role of TXNDC5 in serum starvation-induced proliferation inhibition of HeLa cell.</p><p><b>METHODS</b>TXNDC5 was either over-expressed or knocked down by small interfering RNA (siRNA) in HeLa cells which were then cultured in conventional medium or serum starvation medium. The protein level of TXNDC5 was evaluated by Western blot analysis. The mRNA level of TXNDC5 was measured by quantitative real-time PCR. Cell growth rate was determined by cell proliferation assay kit (MTS method). Cell cycle distribution and apoptosis were detected by flow cytometry.</p><p><b>RESULTS</b>Serum starvation mildly reduced the mRNA level of TXNDC5 (P<0.05), but dramatically increased the protein level of TXNDC5 in HeLa cells. The stability of TXNDC5 mRNA remained unchanged. Cycloheximide abolished the serum starvation-induced up-regulation of TXNDC5 protein. Over-expression of TXNDC5 had no effect on cell proliferation. However, suppression of TXNDC5 attenuated the proliferation inhibition of HeLa cell induced by serum starvation (P<0.05), increased the proportion of cells in S phase (P<0.05), but had no effect on cell apoptosis.</p><p><b>CONCLUSION</b>TXNDC5 mediates serum starvation-induced proliferation inhibition of HeLa cell.</p>

Effects of gestational isoflurane exposure on postnatal memory and learning in rats / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of gestational isoflurane exposure on postnatal memory and learning and growth-associated protein-43 (GAP-43), neuropeptide Y(NPY) expression in the hippocampus of pups.</p><p><b>METHODS</b>Twelve maternal Sprague-Dawley rats at gestation d 18(E18) were randomly divided into isoflurane group (n=6) and control group (n=6). Rats in isoflurane group were exposed to 1.3 % isoflurane for 6 h. For control group, animals breathed in 30 % oxygen and air mixed gas at the same condition. Spatial learning and memory of the offspring were determined with the Morris Water Maze(MWM) after postnatal 4 weeks. The changes of GAP-43 and NPY expression in the hippocampal CA1 region of the pups were determined by immunohistochemistry.</p><p><b>RESULTS</b>In MWM training, the escape latency to platform of the pups in isoflurane group was significantly longer, and the time spent in the third quadrant and times of original platform crossing were less than those of control animals (P<0.05). The number and optical density of GAP-43 and NPY positive neurons in the hippocampus of pups decreased significantly in the isoflurane group compared with the controls (P <0.01).</p><p><b>CONCLUSION</b>Isoflurane exposure in pregnant rats significantly impairs the spatial memory and learning of their pups at a juvenile age, which may be associated with the down-regulation of GAP-43 and NPY in the hippocampus.</p>

Chemical constituets of Sparganium stoloniferum / 中草药

Objective: To study the chemical constituents of Sparganium stoloniferum. Methods: Column chromatographic techniques were applied to isolate the constituents. The chemical structures of the constituents were elucidated on the basis of physicochemical properties and spectral data. Results: Twelve compounds were isolated and identified as β-sitosterol palmitate (1), β-sitosterol (2), azelaic acid (3), docosanoic acid (4), 6,7,10-trihydroxy-8-octadecenoic acid (5), p-hydroxybenzaldehyde (6), sanleng diphenyl-acetypene (7), ferulic acid (8), 3,5-dihydroxy-4-methoxy-benzoic acid (9), 2,7-dihydroxy-xanthone (10), glycerol ferulate (11), and daucosterol (12). Conclusion: Compounds 3,4, and 9-11 are obtained from the plants of Sparganium L. for the first time.

Effects of electroacupuncture and acupoint catgut-embedding on mRNA expression of lipid metabolism gene PPAR-gamma and related lipase of rats with simple obesity / 中国针灸

<p><b>OBJECTIVE</b>To study the mechanism of electroacupuncture and acupoints catgut-embedding in losing weight.</p><p><b>METHODS</b>Simple obesity was induced in male SD rats with high-fat and high-glucose diet for 10 weeks. Thirty-six rats with obesity were randomly divided into model-control group, electroacupuncture (EA) group and catgut-embedding (CE) group, with 12 rats in each group. Another 12 normal SD rats serving as controls (normal control group) were fed by standard rat chow. Rats in the EA group were treated with electroacupuncture at bilateral acupoints of "Housanli" (ST 36), "Tianshu" (ST 25), and "Pishu" (BL 20), once daily for consecutive 15 days. Rats in the CE group were treated with catgut-embedding at the same acupoints with the EA group, once every 7 days. Rats in the other two groups were not interfered. After 15 days of treatment, changes of body weight were observed, the levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in serum were assayed, the activities of lipoprotein lipase (LPL) and hepatic lipase (HL) in liver tissues were measured, and the mRNA expression levels of Peroxisome proliferators activated receptor-gamma (PPAR-gamma) in adipose tissues were determined.</p><p><b>RESULTS</b>After the intervention, body weight and increase of body weight in the EA and CE groups were significantly lower than those in the model-control group (all P<0.01). Compared with the normal control group, there was significant increasing of levels of TC and LDL-C in serum (both P<0.01), descending activities of LPL and HL in liver tissues (P<0.05, P<0.01), and decreased expression levels of PPAR-gamma mRNA in adipose tissues in the model-control group (P<0.05). Compared with the model-control group, there were obviously decreased in TC, LDL-C Level in serum in EA and CE groups (P<0.05, P<0.01), while there was no difference in TG levels among three groups (P>0.05). The activities of LPL and HL were also significantly enhanced in EA and CE groups as compared with those in the model-control group (P<0.01, P<0.05), so were the expression levels of PPAR-gamma mRNA in adipose tissues (both P<0.01).</p><p><b>CONCLUSION</b>Electroacupuncture and acupoint catgut-embedding can regulate the lipid metabolic disorders in rats with obesity through enhancing the expression of PPAR-gamma mRNA in adipose tissues, activating the activities of LPL and HL and decreasing the levels of TC and LDL-C in serum.</p>

CatSper and sperm hyperactivation / 中华男科学杂志

Elevation of sperm Ca2+ seems to be responsible for an asymmetric form of motility called hyperactivation, which is first seen near the time of fertilization. CatSper family proteins are putative cation channels expressed exclusively in the membranes of the sperm flagellum. Hyperactivation requires CatSper proteins, which presumably serve as the route of entry for Ca2+ that operates directly on the flagellar axoneme to increase waveform asymmetry. In this article, the structure of mouse CatSper, the role it plays in sperm hyperactivation and the unsolved problems are described.

The Change of the Level of Plasma D-dimer in Coronary Heart Disease / 医学研究杂志

Objective To study the change of the level of plasma D-dimer(DD) in patients with coronary heart disease(CHD)and its significance.Methods 96 CHD patients were divided into various groups according to coronary angiography and clinical manifestation: 26 patients with simple lesions,47 patients with complex lesions,12 patienty with acute myocardial in-farction(AMI),42 patienty with unstable angina pectoris(UAP),19 patienty with stable angina pectoris(SAP)and 23 patienty with normal healthy control subjects.The level of plasma DD was detected.Results Plasma concentrations of DD were higher in patients with complex lesion((0.501?0.209)mg/L)than in those with simple lesions((0.328?0.1)mg/L)(P

The Localization and Expression of Tyrosine Phosphorylated Proteins During In Vitro Capacitation of Guinea Pig Sperm / 中国生物工程杂志

The aim of this study was to detect the localization and level of tyrosine phosphorylated proteins during in vitro capacitation of guinea pig sperm. Sperm from mature guinea pigs were incubated in modified TALP under 5% CO_2 in air at 37 ℃. The capacitation effect was assessed by chlortetracycline (CTC) staining. Western blotting and indirect immunofluorescence were used to analyze the level and localization of tyrosine phosphorylation. The results showed that guinea pig sperm underwent a time-dependent increase in protein tyrosine phosphorylation during the in vitro capacitation and the percentage of protein tyrosine phosphorylated sperm increased from 36% to 92% from the beginning of incubation to 7h incubation. Also, there was a shift in the site of phosphotyrosine-specific fluorescence from the head of sperm to both the head and the flagellum of sperm. Moreover, there were three proteins phosphorylated in this experiment. After 0 to 0.5h incubation, the protein of 40kDa was detected by anti-phosphotyrosine monoclonal antibody, and the intensity of this protein increased in the following incubation. Then, after 1h incubation, another protein of 80kDa was found and the level of this protein reached the highest point at 3h. Also, in 3h incubation, a protein of 45kDa was detected and the intensity of this protein increased in the following incubation.

Inhibition of mutant-type p53 by a chimeric U6 maxizyme in hepatocellular carcinoma cell lines / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the inhibition of maxizyme (Mz) directed against the mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG --> AGT) both in cell-free system and in MHCC97 cell lines.</p><p><b>METHODS</b>Maxizyme and control mutant maxizyme (G5 --> A5) were designed by computer and cloned into the eukaryotic expression vector pBSKneoU6 (pU6Mz, pU6asMz). Mz was driven by T7 RNA polymerase promoter in vitro. In the cell lines, U6 promoter was driven by RNA PolIII. The mutant type p53 gene fragment was cloned into the pGEM-T vector under the T7 promoter control. The 32P-labeled mtp53 transcript was the target RNA. Cold maxizyme transcripts were incubated with 32P-labeled target RNA in vitro. pU6Mz was introduced into MHCC97 cells by Lipofectamine2000 and mtp53 expression was analyzed by RT-PCR and Western blot.</p><p><b>RESULTS</b>In vitro cleavage showed that pU6Mz was very active with cleavage efficiency of 42% while pU6asMz was not. The wild type p53 was not cleaved. Partial down-regulation of mtp53 mRNA and mtp53 protein were observed in MHCC97 cells transfected with pU6Mz but not those with pU6asMz. The proliferation of MHCC cells was inhibited by MTT analysis.</p><p><b>CONCLUSION</b>Our findings suggest that the chimeric U6 maxizyme against the mtp53 is a new promising gene therapeutic agent in treating hepatocellular carcinoma.</p>

The inhibitory effects on hepatitis B virus replication by stable expression of DN mutants of hepatitis B virus X gene pRev X-GFP / 中华肝脏病杂志

<p><b>OBJECTIVE</b>Persistent replication of hepatitis B virus (HBV) is one of the major obstacles in HBV infection treatment. Reduction or clearance of HBV propagation would be one of the aims of HBV therapy. The drugs approved in clinical used such as nucleotide analogs or interferon, were limited effects on HBV replication. The newly developing gene therapy method, dominant negative mutants, were be used as new promising HBV therapy strategy, and a dominant negative mutant of HBVX gene pRev X-GFP which we have reported in our previous study has some effects both on HBV replication and expression in transient expression, but the effects were interfered by persistent secretion of HBV in HepG2 2.2.15 cells without transfection pRev X-GFP in the experiment. To make sure the effects of dominant negative mutant of pRev X-GFP, we established a HBX DN stable express cell clone, and evaluated the effects of HBX dominant negative mutant on HBV replication.</p><p><b>METHODS</b>The X gene mutant, in which a specific point mutation of 3'-end ATG to AAG and fused with human green fluorescence protein (GFP) were cloned into pRev TRE vector, assigned to pRev HBX-GFP dominant mutant (pRev X-GFP). And the plasmid contains the wild type X gene or GFP gene was cloned into the same vector to construct pRev Xwt, pRev GFP constructs. All the constructs then transfected into HepG2 2.2.15 cells by liposome. After 7 days resistance selection of hygromycin (300 microg/ml), and cell clones which stable expression HBX-GFP, HBXwt, GFP were obtained. After reseeding of 106 cells of each clones in 12 wells with a 12 well cell plate and another 12 wells 2.2.15 cell were serve as blank control. The cells and media were harvested after cultured in DMEM with 10% FBS for 3 days. HBV-related DNA was assayed by dot blot and Southern blot.</p><p><b>RESULTS</b>The 100% expression of pRev HBX-GFP, GFP and wild type X constructs were obtained. The stable expressed HBX-GFP can significantly reduce HBV DNA level both in cell media and cells by dot blot and Southern blot analysis, but not for pRev Xwt and pRev GFP.</p><p><b>CONCLUSION</b>The dominant negative mutant pRev HBX-GFP can significantly inhibit the HBV gene expression. It also suggested that X gene might be one of promising target for HBV gene therapy.</p>

Hammerhead ribozyme against human telomerase catalytic subunit (hTERT) induced apoptosis of liver cancer cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct vector pEGFP-C1-hTERT-ribozyme (pGTRz-U6) and its mutant (pGTmRz-U6) against hTERT containing U6 promoter, then transfect them into human liver cancer cell line SMMC7721 to observe the action of the human telomerase catalytic subunit (hTERT) hammerhead ribozyme on proliferation and apoptosis of human liver cancer cell SMMC7721.</p><p><b>METHODS</b>Eukaryotic expressing vector pGTRz-U6 and mutant pGTmRz-U6 were constructed and transfected into SMMC7721 using Lipofectamine2000 Reagent, with pEGFP-C1 as the control group. After strict screening by G418, positive clones were cultured; the amount of expression of ribozyme and hTERT was detected by RT-PCR; cell proliferation by MTT; telomerase activity by TRAP and silver staining assay; cell apoptosis by FCM.</p><p><b>RESULTS</b>We found that the two ribozymes were expressed persistently in SMMC7721; different expression levels (P < 0.01) of hTERT among SMMC7721-Rz, SMMC7721-mRz and SMMC7721-pEGFP-C1 was exhibited by the analysis of variance with SPSS software. The difference between SMMC7721-Rz and the others is significant in t-test (P < 0.01), while there was no difference between SMMC7721-mRz and SMMC7721-pEGFP-C1 (P > 0.05). With the advance of cell division, telomerase activities of the cells treated by SMMC7721-Rz and SMMC7721-mRz decreased gradually, and the percentage of apoptosis of the cells transfected with Rz and mRz increased gradually. The apoptosis percentage of 7PDS SMMC7721-Rz was 29.86%, while those of SMMC7721-mRz and SMMC7721-pEGFP-C1 were 9.87% and 3.36%, respectively.</p><p><b>CONCLUSION</b>The apoptosis level of SMMC7721 induced by hTERT ribozyme increases as cells divide, and this ribozyme maybe a potential approach for liver cancer gene therapy.</p>

Inhibition of mutant p53 in hepatocellular carcinoma cells by hammerhead ribozyme / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the inhibition of mutant type p53 in hepatocellular carcinoma by hammerhead ribozyme in both cell-free system and MHCC97 cells.</p><p><b>METHODS</b>Hammerhead ribozyme genes (RZ) and control ribozyme (asRZ) directed against mutant p53 (249 codons, AGG --> AGT) were designed by computer. The in vitro transcription plasmid and eukaryotic expression plasmid were constructed into the vector pBSKU6 and pEGFPC1. Human mutant and wild type p53 gene fragment were cloned into the pGEM-T vector under T7 promoter control. In vitro cleavage reaction was carried out by mixing the RZ and target mRNAs which were labeled with [alpha-32P] dUTP. RZ was introduced into MHCC97 cells by LipofectAMINEAM2000 and mtp53 expression was analyzed by RT-PCR.</p><p><b>RESULTS</b>In cell-free systems, RZ showed a specific cleavage activity against mtp53 with cleavage efficiency of 42%, while the wild type p53 was not cleaved. The mRNA level of mtp53 in MHCC97 cells after transfection was reduced by RT-PCR analysis.</p><p><b>CONCLUSION</b>These findings suggest that the hammerhead ribozyme against the mtp53 is a new promosing gene therapeutic agent against hepatocellular carcinoma.</p>