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Objective To observe the safety and clinic effect of umbilical blood stem cell transplantation for the patients with chronic liver failure (CLF).Methods 44 patients with CLF were included in the research and divided into two groups,22 in control group received internal medicine treatment,the other 22 in treatment group received umbilical blood stem cell transplantation in addition to internal medicine treatment.The biochemical index,MELD scores,clinical symptoms,survival situation and adverse reaction of the patients were observed within 2,4,12 and 24 weeks.Results Albumin and prothrombin activity of treatment group were higher than those of control group,the MELD scores of the treatment group was lower than that of control group,the survival rate was higher than the control group,and the difference is significant between the two groups (P ＜ 0.05).There was no significant difference between the two groups in terms of alanine aminotransferase and total bilirubin (P ＞ 0.05).After 4 weeks treatment,fatigue,inappetite,abdominal distention and ascitic fluid of the treatment group were better than that of control group,the difference was statistically significant (P ＜ 0.05).Besides,the patients of the both groups did not have any adverse reaction or hepatocellular carcinoma.Conclusion Umbilical blood stem cell transplantation is safe and effective for the patients with CLF and can improve the survival rate of the patients.
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To study the mechanism of injection of sinomenine (Sinomenine,SIN)into Jiaji point in the treatment of L1-L5 segment Compression of fracture spine (VCF) in rabbits.Methods:54 rabbits were randomly divided into blank control group,model group and sinomenine group (group SIN)by the random number table.All the 2 groups established L1-L5 segment VCF model except the blank control group.After molding,the SIN group was injected SIN at both sides of the L1-L5 section of the vertebral column,the model group and the blank control group were injected the same volume of physiological saline as comparison.The 3 groups were treated for 4 weeks.Fracture of vertebral bone growth differentiation factor 2 (Growth differentiation,FACTOR2,GDF2),vascular endothelial growth factor (Vascular endothelial,growth factor,VEGF) and the expression of VEGF-mRNA were determined by immunohistochemical staining method;in venous blood,activities of nitric oxide (Nitric oxide NO),super oxide dissuade (Super oxide dims,unties,SOD) and bone protection Su (OPG) were detected by ELISA;immunofluorescence was used to measure two chitosan,image analysis method was used to detect spinal bone density in L1-L5 segment,the ELISA was used to detect inflammatory factor interleukin 1 beta (IL-1 beta),IL-6 beta,beta IL-17 in the spinal disc.Results:compared with the model group,in the SIN group,VEGF,VEGFmRNA and GDF2 in L1-L5 segment of spine were highly expressed,and the bone density increased (P＜0.05);two chitosan in intervertebral disc increased,IL-1beta,IL-6 beta,IL-17 beta,(P＜ 0.05);NO in venous blood decreased OPG,SOD increased (P＜0.05),the differences were statistically significant.Conclusion:injection of SIN into Jiaji can promote the healing of VCF in L1-L5 segment in rabbits.
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Objective:To reveal the action mechanism and efficacy of electroacupuncture (EA) plus external application of Wei Ling Xian (Radix Clematidis) extract in treating knee osteoarthritis (KOA), for providing theoretical evidence and novel plan for the treatment of KOA.Methods:Totally 284 inpatients were divided into an EA group and an observation group by the random number table, 142 cases in each group. The EA group was intervened by EA, ultrashort waves, and oral administration of Glucosamine Hydrochloride capsules; while the observation group was by external application of Wei Ling Xian (Radix Clematidis) extract at topical area in addition to the treatment given to the EA group. The intervention lasted 14 d in both groups. X-ray examination of knee joint was ordered before and after treatment, as well as the detection of positive expression of matrix metalloproteinase-1 (MMP-1) in cartilage of knee joint, hyaluronic acid (HA) and interleukin-1β (IL-1β) in the inflammatory effusion; the Western Ontario and McMaster Universities osteoarthritis index (WOMAC) and visual analogue scale (VAS) were adopted to evaluate the clinical efficacy.Results:In the observation group, the X-ray examination result of knee joint was significantly improved, the positive expressions of MMP-1 and IL-1β content were significantly decreased, the level of HA was increased, WOMAC and VAS scores dropped, and the total effective rate was 91.5%, which were significantly different from those in the EA group (P<0.05).Conclusion:EA plus external application of Wei Ling Xian (Radix Clematidis) extract can produce a more significant efficacy in treating KOA compared to ordinary EA treatment.
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Objective To investigate the therapeutic effect of Guizhi-Fuling pill combined with danazol for endometriosis and its possible therapeutic mechanisms. Methods A total of 316 patients with endometriosis were recruited and randomly divided into a routine treatment group and a combined treatment group, 158 in each group. The conventional treatment group were treated by oral danazol tablets, and the combined treatment group by oral danazol and Guizhi-Fuling pill for 28 days. The serum levels of vascular endothelial growth factor (VEGF), VEGF receptor Flk-1, estrogen receptorα(ERα), 17β-estradiol (E2), IL-2, IL-6 and IL-8 were detected by the enzyme-linked immunosorbent assay. The expressions of VEGF and Flk-1 mRNAs in the ectopic endometriotic stromal cells were determined with the reverse transcriptase-polymerase chain reaction. Results The total effective rate in the combined treatment group was significantly higher than that in the routine treatment group (93.7% vs. 78.5%; χ2=13.968, P<0.01), and the recurrence rate was significantly lower (5.4%vs. 13.7%;χ2=19.984, P<0.01). After the treatment, the serum levels of VEGF (2.93 ± 0.07 pg/ml vs. 6.85 ± 0.12 pg/ml;t=354.679, P<0.01), Flk-1 (2.97 ± 0.12 pg/ml vs. 5.87 ± 0.17 pg/ml;t=175.179, P<0.01), ERα(4.93 ± 0.29 pg/ml vs. 6.85 ± 0.37 pg/ml;t=51.337, P<0.01), E2 (11.69±1.09 pg/ml vs. 14.10 ± 0.78 pg/ml;t=22.601, P<0.01), IL-2 (2.27 ± 0.12 pg/ml vs. 2.31 ± 0.16 pg/ml;t=2.514, P<0.05), IL-6 (0.39 ± 0.03 pg/ml vs. 0.59 ± 0.06 pg/ml;t=37.476, P<0.01) and IL-8 (0.47 ± 0.04 pg/ml vs. 0.81 ± 0.09 pg/ml;t=43.393, P<0.01) in the combined treatment group were significantly lower than those in the routine treatment group;the expressions of VEGF (0.69 ± 0.08 vs. 2.11 ± 0.09; t=148.229, P<0.01) and Flk-1 (4.67 ± 0.14 vs. 5.61 ± 0.22;t=45.311, P<0.01) mRNAs in the ectopic endometriotic stromal cells in the combined treatment group were significantly lower than those in the routine treatment group. Conclusions Guizhi-Fuling pill combined with danazol can downregulate the expressions of VEGF and Flk-1 in the ectopic endometriotic stromal cells, reduce the serum levels of VEGF, Flk-1, ERαand E2, alleviate inflammation, and improve symptoms.
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Objective To observe the effect ofDanhuai-Yinxie condensed pill on the expression of VEGF and its receptors in the model rabbits with psoriasis.Methods Fourty-two healthy Japanese white rabbits were randomly divided into the control group, the model group, the Acitretin group, and the low-, medium- and high-doses ofDanhuai-Yinxie condensed pill groups. The backs of rabbits’ ears were wiped with 5% propranolol for 4 weeks in the latter four groups, while with saline for the same period in the control group. The rabbits in the Acitretin group were intragastric administrated with 1% Acitretin one day since the models of psoriasis were successed, while the rabbits in the low-, medium- and high-doses ofDanhuai-Yinxie condensed pill groups were intragastric administrated withDanhuai-Yinxie suspension 20, 40, 80 mg/kg daily, respectively. The rabbits in the control and model groups were intragastric administrated with equal-volume saline daily. The expression of VEGF, its receptor 1 (FLT-1) and receptor 2 (KDR) were detected by ELISA method, and the expression of VEGF mRNA was detected by PCR method, dermal papillary vascular density (MVD) and the T lymphocytes proliferation from peripheral blood were detected.Results The expression of VEGF (114.43 ± 952 ng/ml, 109.65 ± 9.15 ng/mlvs. 179.90 ± 11.30 ng/ml), VEGF mRNA (0.83 ± 0.07, 0.80 ± 0.06vs. 2.74 ± 0.15), FLT-1 (4.54 ± 0.26 ng/ml, 4.16 ± 0.24 ng/mlvs. 6.92 ± 0.38 ng/ml), KDR (2.48 ± 0.16 ng/ml, 2.51 ± 0.19 ng/mlvs. 6.79 ± 0.27 ng/ml), MVD (10.12 ± 1.97, 9.83 ± 1.12vs. 16.45 ± 2.05) and T lymphocytes proliferation (87.35% ± 6.29%, 86.21% ± 6.42%vs. 123.74% ± 9.18%) in the medium- and the high-dose groups significant decreased than those in the model group (P<0.05).ConclusionDanhuai-Yinxiecondensed pill could reduce the expression of VEGF, block the binding of VEGF to its receptors, inhibit angiogenesis, inhibit the reconstruction of subcutaneous tissues, reduce the dysplasia of dermal microvessels, and inhibit the proliferation of T cells.
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ObjectiveTo detect the effect of electroacupuncture combined with acupoint injection of deer melon polypeptide on the bone morpho-genetic protein synovial, BMPs and serum rheumatoid factor IgA-RF, IgM-RF.MethodsA total of 246 patients with Rheumatoid arthritis were divided randomly into control group and combined treatment group, 123 patients in each group. The control group received the treatment of electro-acupuncture, drug and Ultrashort wave therapy, and the combined treatment group continue to receive acupoint injection of deer melon polypeptide after 30 minutes when the treatment of control group was completed. Both groups were treated for 2 weeks. The expression of BMPs, MMP-1 and the ESR, serum rheumatoid factor IgA-RF, IgM-RF were detected. The morning stiffness time, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and visual analogue scale (VAS) were recorded for outcome assessment.ResultsCompared with control group after treatment, the positive expression rates of ESR (8.79 ±1.61 mm/hvs.12.41 ± 2.57 mm/h,t=3.654), IgM-RF (22.38% ± 5.45%vs.39.57% ± 6.18%,t=2.365), IgA RF (32.84% ± 6.57%vs.49.84% ± 4.20%,t=2.574) in the combined treatment group significantly decreased. Compared with the control group, the expression of BMPs (15.43 ± 2.94vs.7.22 ± 1.52,t=3.914) in the combined treatment group significantly increased, while MMP-1 (8.01% ± 1.54%vs. 15.34% ± 2.47%,t=2.015) in the combined treatment group significantly decreased (P<0.05). Compared with control group, the outcomes VAS (3.37 ± 0.35 vs.5.75 ± 0.39, t=3.251), WOMAC (22.36 ± 3.49vs.30.50 ± 4.11,t=4.021), and morning stiffness (34.32 ± 5.57 minvs. 49.84 ± 4.20 min,t=3.914) in the combined treatment group significantly decreased (P<0.05). The total effect rate (82.11%vs. 71.55%,χ2=12.764) of combined treatment group was significantly higher than control group (P<0.05).Conclusion Electroacupuncture combined with acupoint injection of deer and melon peptide can improve effect rate.
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OBJECTIVE@#To investigate the correlation between cyclin-dependent kinase inhibitor p27kip1 and trastuzumab-resistance in gastric cancer. @*METHODS@#We selected HER2-overexpressed human gastric cancer cell line NCI-N87 to establish trastuzumab-resistant NCI-N87/TR cell line by stepwise exposure to different doses of trastuzumab. The 50% inhibitory concentration (IC(50)) of trastuzumab and resistance index (RI) were calculated or analyzed by MTT assay. The expression levels of cdk2 and p27kip1 were detected by Western blot. After the treatment with cdk2 inhibitor (Purvalanol A), the expression levels of relevant proteins in NCI-N87/TR cells were detected by Western blot, and the sensitivity to trastuzumab was analyzed by MTT assay. @*RESULTS@#Compared with NCI-N87 cells, the expression of cdk2 was significantly increased in NCI-N87/TR cells (P<0.001), while the expression of p27kip1 showed a significant decrease (P<0.001). Restoration of the p27kip1 protein expression by cdk2 inhibitor (Purvalanol A) increased the sensitivity of NCI-N87/TR to trastuzumab. @*CONCLUSION@#Down-regulation of p27kip1 might be a mechanism for triggering trastuzumab resistance to gastric cancer cell line NCI-N87.
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Humans , Antineoplastic Agents , Pharmacology , Cell Line, Tumor , Cyclin-Dependent Kinase 2 , Genetics , Metabolism , Cyclin-Dependent Kinase Inhibitor p27 , Genetics , Metabolism , Drug Resistance, Neoplasm , Purines , Pharmacology , Stomach Neoplasms , Genetics , Metabolism , Pathology , Trastuzumab , PharmacologyABSTRACT
Objective To observe the effect ofShenmal injection on vascular endothelial function after cerebral ischemia reperfusion in rats.Methods 30 rats were randomly divided into a sham operation group, a model group and aShenmal group, with 10 in each. Except the sham operation group, animal models of cerebral ischemia reperfusion injury were established by the right internal carotid artery occlusion of middle cerebral artery. Then shenmal injection was given to theShenmal group, 2 ml/100 g Sodium Chloride was given to the other groups, once per day, for successive 7 days. Endothelin-1 (ET-1), nitric oxide (NO), nitric oxide synthase (NOS), serum high sensitivity C-reactive protein (hs-CRP), and homocysteine (Hcy) were tested. Results In Shenmal group, ET-1(126.10 ± 6.07 ng/mlvs.105.41 ± 0.09 ng/ml), hs-CRP (15.93 ± 2.79 mg/Lvs. 9.33 ± 1.32 mg/L) and Hcy (11.01 ± 0.98 μmol/Lvs. 8.13 ± 0.46 μmol/L) were decreased after the treatment, while NOS (168.87 ± 9.25 U/mlvs.242.25 ± 10.36 U/ml) and NO (84.03± 6.02vs.102.42± 5.05) were increased after the treatment with statistical difference(P<0.05).ConclusionShenmalinjection can improve NOS activity and promote the synthesis of NO, decrease the level of plasma ET-1, hs-CRP and Hcy, reduce endothelial inflammatory exudation, and prevent vascular endothelial dysfunction caused by cerebral ischemia reperfusion injury.
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Objective To observe the protective effects of Cordyceps Extract (Cordyceps sinensis, CS) on ischemia reperfusion injury of transplanted kidney in renal allograft rejection, and to investigate the inhibition of immune regulation mechanism.Methods 100 rats were randomly divided into a blank control group, an ischemia reperfusion group, a cyclosporine A group, a Cordyceps high dose group (CSA), and a Cordyceps small dose group (CSB). Except the blank control group, all the other groups were underwent same race variant body left renal transplantation. 2 hours after the transplantation, cyclosporin A 2.0 mg/(kg?d) was injected to the cyclosporine group, CSA group, and CSB group. 1% CS of 5 and 10 ml/kg by gavage was respectively given to CSA and CSB groups. 10ml/kg sodium chloride was injected to the bland control group and the ischemia reperfusion group. After 14 days (d) of administration, the content of blood urea nitrogen (BUN), Cystatin C (Cys C), creatinine (Cr), total superoxide dismutase (T-SOD), serum malondialdehyde(MDA), lactate dehydrogenase (LDH), serum total protein (TP), albumin (ALB) were detected. Results Compared with the cyclosporin A group, the content of BUN(8.71 ± 0.87mmol/L, 9.78 ± 1.01mmol/Lvs.12.59 ± 1.54mmol/L), Cys C(1.36 ± 0.09 mg/L, 1.40 ± 0.11mg/L vs.1.94 ± 0.12 mg/L), Cr(117.79 ± 15.35μmol/L, 121.28 ± 16.01μmol/L vs.133.76 ± 17.07 μmol/L), LDH(157.32 ± 20.93U/L, 154.35 ± 19.39U/L vs.187.78 ± 25.18 U/L), MDA(122.23 ± 19.41nmol/ml, 125.29 ± 25.97nmol/ml vs.184.23 ± 26.74 nmol/ml) were decreased in the CSA group and CSB group, T-SOD(80.35 ± 7.71U/ml, 78.30 ± 7.79U/ml vs.56.43 ± 7.25 U/ml) was increased(P<0.05).Conclusion CS can significantly inhibit lipid peroxidation, renal ischemia reperfusion caused a decrease in MDA and LDH production, increase the activity of T-SOD and TP, ALB, reduce the damage of kidney function, have a dual immunomodulatory effects obvious, with cyclosporin A anti rejection has synergistic effects, while increasing the body immunity, to allograft renal transplantation has protective effect of renal ischemia / reperfusion injury in rats.
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Objective To investigate the clinical efficacy of umbilical blood stem cell transplantation (UCBSCT) on the treatment of hepatitis B liver cirrhosis. Methods Forty-eight patients with hepatitis B liver cirrhosis were enrolled and divided into the treatment group and the control group. There were 25 patients in the treatment group , who received UCBSCT treatment based on conventional liver protection treatment and 23 patients in the control group , who received conventional liver protection treatment. The changes of liver function , coagulation function, clinical symptoms, signs and side effects were studied before the treatment and at 2, 4, 12 and 24 weeks post-treatment. Results The levels of albumin, cholinesterase, and prothrombin activity in the treatment group were higher than those before treatment and were higher than those in the control group. The parameters in the control group were not significantly changed before and after the treatment (P > 0.05). The levels of alanine aminotransferase,aspartate aminotransferase,total bilirubin in both two groups were not significantly changed before and after the treatment (P > 0.05). After 4-week treatment,the differences on improvement of appetite , lacking in strength , abdominal distension , ascites were statistically significant in the treatment group compared with the control group (P < 0.05). No adverse reactions were observed in all groups. Conclusion UCBSCT on the treatment of hepatitis B liver cirrhosis is safe and reliable.
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Objective To investigate the effect of electro-acupuncture at Guanyuanshu, Xinshu, Neiguan points on cardiac function after acute myocardial ischemia in rabbits. Methods A total of 36 rabbits were randomly divided into a control group, a model group and a treatment group. Acute myocardial ischemia was induced with Jugular vein injection of pituitrin. At 10 min before modeling, 1 hour and 1 week after modeling, the levels of malondialdehyde (MDA) and creatine kinase(CK) were determined respectively, the cardiac function was assessed as the maximum rate of left ventricular pressure rise(dp/dtmax)and the peak left ventricular pressure (LVSP). Results After 1 week of treatment, the serum levels of CK and MDA in the treatment group reduced significantly(120.21±10.67 U/L, 21.35±2.04 mmol/L], compared to the model group (162.37±13.21 U/L, 30.13±2.75 mmol/L) (all P<0.05);the dp/dtmax and LVSP decreased percentage in the treatment group (20%±2.43%, 7.22%±0.97%) decrease significantly compared to the model group (31.5%± 4.14%, 12.19%±2.21%) (all P<0.05). Conclusion Electro-acupuncture of Guanyuanshu, Xinshu, Neiguan acupoints can significantly improve cardiac function after acute myocardial ischemia and protect against acute myocardial ischemia in rabbits.
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Objective To observe the Cordyceps extract on experimental rats with diabetic kidney and to explore its mechanism of action.Methods The rat models of diabetic were established by intraperitoneal injecting streptozotocin.Kidney weight and the index of kidney were measured,and 1,4,7weeks blood sugar,serum creatinine,urine microalbumin and urea were dynamically observed for evaluating Cordyceps extract on experimental rats with diabetic kidney.Results The 1,4,and 7 weeks of kidney weight was (0.662± 0.062) g,(0.670±0.061) g,(0.657±0.063) g,renal index was (0.312±0.041) ％,(0.309±0.402) ％,(0.311 ±0.0317) ％,serum creatinine was (0.51±0.03)mg/d1,(0.57±0.03)mg/d1,(0.52±0.02)mg/d1,albumin was (1.77±0.17)mg/24 h,(1.52±0.19)mg/24 h,(1.56±0.11)mg/24 h.urea was (90.71 ±0.37)mmol/L,(91.57±0.48) mmol/L,(90.56±0.39)mmol/L in the normal control group respectively.While in the model group,the value of kidney weight was (0.879±0.037)g,(0.912±0.038)g,(0.871±0.393)g,renal index was (0.494±0.039)％,(0.487±0.038)％,(0.465±0.235)％,serum creatinine was (3.71 ±0.14) mg/d1,(3.51±0.12) mg/d1,(3.32±0.11)mg/d1,urinary albumin was (7.29±0.22)mg/24 h,(7.11±0.34)mg/24 h,(7.14±0.21)mg/24 h.urea was (109.59± 8.42),(92.52±2.41),(90.71 ±0.67) respectively.Compared with the normal control group these values were decreased significantly in the model group (P＜0.05).In the Cordyceps sinensis group kidney weight was (0.837±0.036)g,(0.747±0.029)g,(0.694±0.357)g,renal index was (0.412±0.024)％,(0.395±0.037)％,(0.361±0.027)％,serum creatinine was (1.44±0.11)mg/d1,(0.81± 0.04) mg/d1,(0.62±0.03) mg/d1,urinaryalbumin was (5.71±0.25)mg,(4.52±0.21)mg,(3.96±0.15)mg,urea was (109.59± 8.42) mmol/L,(92.52±2.41) mmol/L,(90.71 ± 0.67) mmol/L respectively.Compared with the model group these values were decreased significantly(P＜0.05) in the Cordyceps sinensis group.Conclusion Cordyceps sinensis extract has a protective effect on kidney of diabetic rats.
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Objective To investigate the prevalence and charateristics of carotid atherosclerosis among healthy middle-aged people of community. Methods Totally 1930 subjects aged 50 years and above were selected randomly from 10027 participants recruited during Phase 3 of the Guangzhou Biobank Cohort Study (GBCS).Common carotid artery intima-media thickness (CCA-IMT) and carotid plaques (CP) were measured by B-mode ultrasonography using ALT HDI 3000 mainframe with a high-resolution. Results The prevalences of CCA-IMT and CP in men were higher than women [41.5％(400/963)vs.16.2 (157/967),34.1％ (328/967) vs.13.8％ (133/967),both P＜0.001].The prevalence of CCA-1MT was higher with age increasing in either men or women in groups of 50-yrs,60-yrs and 70-79 yrs (all P＜0.001).The most of CP was cankerous spot (37.8％),secondly hard spot (24.9％),soft spot (22.1％) and the prevalence of flat spot was lowest (15.2％).The number of CP was not significantly different between the left and right side (51.0％ and 49.0％).The most common location of CP was bifurcations of carotid arteries (61.7 ％),next the common carotid artery (21.0％) and internal carotid artery (ICA) was lowest (17.3 ％).The prevalence of CP was 44.4％ (83/87) in women aged 50- yrs,it was higher than that in men 20.9％ (108/517).However,the prevalence of CP was 79.1％ (409/517) in men aged more than 60 yrs,higher than that in women (55.6％). Conclusions The carotid atherosclerosis is commonly encountered disease in the male elderly,and the most of CP was cankerous spot in bifurcations of carotid arteries
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BACKGROUND: Streptococcus mutans (S. mutans) is generally considered to be the principal aetiological agent for dental caries, thrS gene may relate to the virulence of S. mutans involved in the adherence, acidogenicity and acidodurance.OBJECTIVE: To investigate the conservation status of the thrS gene of S. mutans and to construct the homologous recombinant plasmid.METHODS: Southern Blot was used to analyze the distdbuUon of thrS gene in S. mutans. The upstream and downstream sequences of thrS gene were cloned respectively into multiple cloning sites of suicide plasmid pFW5 to construct the recombinant plasmid,RESULTS AND CONCLUSION: The thrS gene was conserved in 6 strains of S. mutans in this test. By PCR analysis and enzyme digesting, it was confirmed that S. mutans thrS gene homologous recombinant plasmid was successfully constructed,which can be used in future research of construction of thrS -negative mutans of S. mutans strain UA159.
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Objective:To explore avirulent strain-specific new genes or new functions of already known genes from Streptococcus mutans of serotype c.Methods:Twenty-six DNA fragments unique to avirulent strain of Streptococcus mutans were sequenced.The sequences of these presumptive avirulence DNA fragments were subjected to homology search through BLASTn and BLASTx in public database,and their putative biological functions were analyzed.Results:The size of the DNA fragments ranged from 113 bp to 776 bp.The average G+C content was 38.27%,similar to that of the gene-codingsequences in Streptococcus mutans strain UA159 whose genome sequences were just completed.Seven clones were picked repeatedly.Of the nineteen DNA fragments,eight potentially represented new DNA fragments were registered in GenBank.The remaining DNA fragments showed high homology to known genes of Streptococcus mutans strain UA159.Conclusions: The gene analysis and identification supply the groundwork for further study of gene functions of the avirulent strain of Streptococcus mutans serotype c.
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Objective:To knock the phospho-sugar mutase gene (psm) out of the genome of S. mutans strain UA159 and to construct the mutant will lay a foundation for the study of the function of the psm. Methods:Two upstream and downstream DNA sequences of the psm were selected and cloned respectively into multiple cloning sites I and II of suicide plasmid pFW5 to construct the recombinant plasmid which was confirmed by enzyme digesting and sequencing. The recombinant plasmid was transformed into S. mutans UA159 by natural transformation and antibiotic was used to screen the positive transformants. According to the principle of homologous recombination, allelic exchange between the recombinant plasmid and S. mutans UA159 was achieved. Results:By PCR analysis and sequencing, it was confirmed that the psm of S. mutans UA159 was substituted for resistant gene of spectinomycin. Conclusion: The psm-negative mutants of S. mutans UA159 is successfully constructed.
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Objective:To construct a suppression subtractive library of suppression-related genes from c serotype Streptococcus mutans (S.mutans). Methods:After being isolated from virulent and avirulent strain of S. mutans respectively, the intact and high-pure genomic DNAs were digested with restriction enzyme AluⅠ. The digested DNA of the avirulent strain ligated with an adaptor was used as tester DNA, and that of the virulent strain as driver DNA. Then the suppression subtractive hybridization(SSH) was carried out, the efficiency of ligation and subtraction were detected respectively. The subtracted fragments were inserted into vector pCR2.1 using T/A cloning kit and transformed into E. coli TOP10F' competent cells. The white colonies were selected to construct the suppression subtractive library. Results: Through electrophoresis of AluⅠ-digested DNAs, a smear ranged from 0.1 to 2.0 kb was observed. The ligation efficiency of tester DNA with adaptor was at least higher than 25 percent. The subtraction efficiency confirmed the success in enrichment of differential genes between virulent and avirulent strain of S. mutans. In the subtracted group, the appearance time of the 23S rRNA gene in both tester and driver DNA was later than that in the unsubtracted group by twelve cycles. It suggested that suppression subtractive hybridization happened indeed. Then the subtracted fragments were cloned and the suppression-related gene library between virulent and avirulent strain of S. mutans was constructed. Conclusions:The suppression subtracted library of suppression-related genes has been constructed.
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Objective:To construct the prokaryotic expression vector pET28a(+)-comE, to get the highest expression of comE in E. coli BL21(DE3), and to obtain the purified ComE fusion protein. Methods: ComE gene was amplified by PCR with specific primers from genome of Streptococcus mutans UA159. After digested by two restriction endonucleases BamH Ⅰand Xho Ⅰ, the gene segment was inserted into vector pET28a(+). Then the recombinant vector was transformed into E. coli BL21(DE3). The protein expression was induced by IPTG and the result was confirmed by Western blot. The solubility of the fusion protein was examined by 12% SDS-PAGE, and the expression conditions were optimized. Finally the fusion protein was purified by a two-step purification procedure utilizing Ni2+ chelating column and S75 size exclusion column. Results: The recombinant plasmid was confirmed by PCR, restriction endonucleases digestion and sequence analysis, which showed that the inserted segment was identical to comE gene reported by GenBank without nucleotide mutation. The fusion protein which was confirmed by Western blot can be expressed in E. coli BL21(DE3) and existed both in supernatant and precipitation. The maximum expression product amount was obtained when the A600 value of E. coli BL21(DE3) was 0.4, IPTG concentration was 0.10 mmol/L and induction time was 4 h. Conclusion: The recombinant plasmid is constructed and the ComE fusion protein is purified by the optimum conditions.