Callus of Gentiana crassicaulis was initiated from hypocotyl and cotyledon on MS medium supplemented with 2mg/L 2.4-D and 0.5 mg/L BA.Induction frequencies of callus were 100%.Somatic embryos were formed on MS medium containing 2mg/L BA,3mg/L ZT, 1mg/L NAA,3mg/L GA,three time MS organics and vitamins,6% sucrose and 500 mg/L LH and three time FeSO4 (Na2EDTA) .40% Callus produced somatic embryos.It developed into complete plants on hormon-free MS medium.

Objective To investigate the curative effects and adverse reactions of pure radiotherapy and concurrent chemotherapy and radiotherapy for patients with cervical cancer.Methods One hundred and twenty-seven patients with cervical cancer who accepted treatment in the Affliated Hospital of Inner Mongolia Medical Unive.rsity from May 2010 to May 2012 were collected.All patients were divided into two groups:pure radiotherapy group (n =65) and concurrent chemotherapy and radiotherapy group (n =62).The curative effects,adverse reactions and survival of two groups were observed.Results All patients were completed treatment.The median follow-up time was 42 months.The rate of complete response in the pure radiotherapy group was 80.0％ (52/65),and the rate in the concurrent chemotherapy and radiotherapy group was 82.26％ (51/62),with no significant difference (x2 =1.22,P =0.352).The 1-year overall survival rates in the pure radiotherapy group and the concurrent chemotherapy and radiotherapy group were 95.38％ and 95.16％ respectively,with no significant difference (x2 =0.32,P =0.533),but the 3-year overall survival rates were 81.54％ and 90.32％ respectively,the 5-year overall survival rates were 72.31％ and 83.87％ respectively,with significant differences (x2 =5.09,P =0.015;x2=3.87,P =0.039).However,for the patients who were ≥ 60 years,the 1-year overall survival rates in the two groups were 94.62％ and 93.91％ respectively,the 3-year overall survival rates were 85.02％ and 87.25％ respectively,the 5-year overall survival rates were 70.06％ and 73.58％ respectively,with no significant differences (x2 =0.06,P =0.753;x2 =1.16,P =0.279;x2 =0.48,P =0.511).The adverse reactions were mainly in grades 1-2.There were significant differences in the rates of leucopenia (56.10％ vs.72.20％),thrombocytopenia (58.82％ vs.76.80％),nausea and vomiting (34.04％ vs.56.90％) among the two treatment groups (x2 =11.23,P =0.003;x2 =11.82,P=0.002;x2 =12.77,P =0.000).Conclusion The curative effect of concurrent chemotherapy and radiotherapy is better than that with pure radiotherapy for patients with cervical cancer,which can improve the 3-year and 5-year overall survival.But at the same time,it should be noted that the rates of adverse reactions may be increased during the same period.For the age of 60 or more patients with cervical cancer,concurrent chemotherapy and radiotherapy does not achieve even greater survival benefit.

Objective The aim of this study is to investigate the expression of Survivin in laryngeal car-cinoma and to elucidate the relationship between cell proliferation and the expression of Survivin .Methods The expression and DNA quantification of Survivin in 63 cases of laryngeal carcinoma and 15 cases of normal laryngeal tissues were detected by immunohistochemical staining ( SP) and flow cytometry .Results Forty-five sections of laryngeal carcinoma were positive for survivin as determined by immunohistochemical staining ,but Survivin was undetected in normal laryngeal tissues .The intensity of Survivin expression was significantly increased with tumor differentiation and lymph node metastasis (P<0.05),and was negatively correlated with age ,gender and clinical stage of patients(P>0.05).High DNA index(DI)and proliferation index(PI)were detected in laryngeal carcino-ma(P<0.05),and PI was positively correlated with expression of Survivin (P<0.05).Conclusion Survivin overexpression triggers laryngeal carcinoma cell hyperproliferation ,especially in development of laryngeal carcino-ma.These data identify Survivin as an important target in laryngeal carcinoma and provide a translational pathway for developing new therapies against this target .

Objective To investigate the relationship between plasma homocysteine (Hcy), Kv1.3 channel and cardiac troponinI (cTnI) in patients with acute ST-segment elevation myocardial infarction (STEMI). Methods According to the level of Hcy, 80 STEMI patients were divided into STEMI with Hhcy group (Hcy > 15 μmol/L, n=41) and control group (STEMI group, Hcy≤15μmol/L, n=39). The Hcy, blood lipid and cTnI were detected with automatic biochemistry analyzer, respectively. Peripheral lymphocytes were isolated by ficoll density gradient centrifugation. Real-time PCR was used to detect mRNA expression of Kv1.3, and Western blot assay was used to detect protein expression of Kv1.3. Results cTnI concentrations were obviously higher in STEMI with Hhcy group than those in STEMI group (μg/L:22.997 ± 5.880 vs. 12.881 ± 6.343;P<0.01). Multiple linear regression analysis showed that age, gender, hypertension, diabetes mellitus, smoking, family history, total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) had no obvious influence on Hcy (P>0.05). The relative expression levels of Kv1.3 mRNA and protein were significantly higher in STEMI with Hhcy group (1.35±0.14, 0.85±0.12) than those in STEMI group (1.00 ± 0.07, 0.64 ± 0.05, P<0.05). Moreover, there was a positive relation between Hcy level and the mRNA and proteinexpression of Kv1.3 channel (r=0.299, r=0.542, P<0.05). There was a positive relation between protein expression levels of Kv1.3 channel and cTnI (r=0.644, P<0.05). Conclusion Our results support that Hcy could exacerbate the concentration of cTnI through playing an important role in the Kv1.3 mRNA and protein expression in lymphocytes.

Objective To observe the influence of different level of hyperhomocysteinemia on mRNA and protein expressions of KV1 .3 ,CaN,NFAT,IL-6 and TNF-αin lymphocytes of patients with acute ST-segment elevation myocardial infarction (STEMI).Methods We selected 90 STEMI patients and divided them into three groups according to the level of plasma homocysteine:the first experimental group (STEMI group,Hcy<1 5μmol/L, n=30),the second experimental group (STEMI with mild Hhcy group,Hcy 15~30μmol/L,n=30)and the third experimental group (STEMI with intermediate Hhcy group,Hcy>30 μmol/L,n=30 ).Another 30 healthy examined people were selected as control group (n=3 0 ).Peripheral lymphocytes were isolated by Ficoll density gradient centrifugation.The Hcy in the plasma was measured with the IMX assays.Real-time quantitative PCR (RT-PCR)was used to detect mRNA expressions of KV1.3,CnAα,NFAT1,IL-6 and TNF-αand Western blot technique was used to detect the expressions of KV1.3,CnAαand NFAT1.Results The mRNA and protein expression levels of KV1.3,CnAαand NFAT1 in each experimental group were significantly higher than those in control group (P<0 .0 5 or P<0 .0 1 ).Multiple comparison in each experimental group showed that compared with that in the first experimental group,the expression level of the second experimental group increased (P<0.05 or P<0.01)and compared with first and second experimental groups,the expression level of the third experimental group increased (P<0.05 or P<0.01).The mRNA expression levels of IL-6 and TNF-αin each experimental group were significantly higher than those in control group (P<0.05 or P<0.01 ).Multiple comparison in each experimental group showed that compared with that in the first experimental group,the expression level of the second experimental group increased (P<0 .0 5 or P<0 .0 1 )and compared with first and second experimental groups,the expression level of the third experimental group increased (P<0.01).Plasma total Hcy levels were positively correlated with mRNA and protein expressions of KV1.3 in all observed groups (r=0.503 P=0.000,r=0.726 P=0.000).Conclusion The higher level of Hcy in plasma,the higher mRNA and protein expression levels of KV1.3,CnAα,NFAT1 and the higher mRNA expression levels of IL-6,TNF-αin the lymphocyte of STEMI patients,which may be one mechanism for Hcy exacerbating the inflammatory reaction of STEMI.

Objective To observe the effects of different concentrations of SB203580, the inhibitor of P38MAPK, in process of high glucose (GS)-induced renal tubular epithelial-myofibroblast transdifferentiation (TEMT). Methods The cultured human renal tubular epithelial cells (HK-2) were divided into control group (5.5 mmol/L GS), GS (30 mmol/L GS) group and different concentrations of SB203580 (30 mmol/L GS +5, 10, 20 and 30 μmol/L SB203580) groups. The treat?ments were for 48 hours. MTT assay was used to observe cell proliferation. The median inhibiting concentration (IC50) was cal?culated. Western blot assay was used to detect the expressions of P38MAPK, P-P38MAPK andα-smooth muscle actin (α-SMA) in control group, high-glucose group and S30 group. The expression ofα-SMA was also detected by the method of im?munofluorescence. Results 1.Compared with control group, there was no significant inhitory effect on proliferation rate in DMSO group (P>0.05). There were increased HK-2 cells in high glucose group and S5group (P<0.05). Proliferation rates were significantly decreased in S20 and S30 groups (P<0.05). Compared with high glucose group, the proliferation rates of HK-2 cells were inhibited in S5, S10, S20 and S30 groups (P<0.05). 2. The expression of P-P38MAPK was significantly higher in high glucose group and S30 group than that of control group (P<0.05). Compared with high glucose group, the ex?pression of P-P38MAPK was significantly decreased in S30 group (P<0.05), whereas no significant difference in the expres?sion of P38MAPK between the two groups (P>0.05). 3. Compared with control group, the expression ofα-SMA was signifi?cantly increased in high glucose group and S30 group (P<0.05). Compared with high glucose group, the expression of α-SMA was significantly decreased in S30 group (P < 0.05). Conclusion The 30 mmol/L GS can lead to TEMT in HK-2 cells. The more suitable inhibitory concentration of SB203580 in the process of TEMT is 30μmol/L. SB203580 can slow down the process of TEMT by inhibiting P38MAPK activation and inhibiting proliferation and the expression ofα-SAM s of HK-2 cells.

Objective To explore the effect and the possible pathway of different concentrations of QLT0267,which was the inhibitor of the integrin-linked kinase (ILK),on the process of high glucose-induced tubularepithelial-myofibroblast transdifferentiation (TEMT) in human renal tubular epithelial cells (HK-2).Methods HK-2 cells were exposed to 30 mmol/L GS,and TEMT model was established.After excluding the effect of high osmotic in TEMT,HK-2 cells were divided into 6 groups by different concentrations of GS and QLT0267 for 48 hours.The rate of the cell proliferation was calculated by MTT.The expression of ILK and α-smooth muscle actin (α-SMA) were determined by immunofluorescence and Western blot,and the expression of protein kinase B (AKT),phosphorylated protein kinase B (p-AKT),and E-cadherin were determined by Western blot.Results (1) The expression of ILK,p-AKT,and α-SMA in HK-2 cells were unregulated and the expression of E-cadherin was downregulated for 48 hours with glucose treating vs control (P ＜ 0.05);(2) The proliferation rate in high glucose group was higher than the group which concentration of QLT0267 was greater than 5 μmol/L (P ＜ 0.05);(3) With the concentrations of QLT0267 increased,the expression of p-AKT,α-SMA was gradually decreased (all P ＜ 0.05),and the expression of E-cadherin was gradually increased (all P ＜ 0.05).Conclusions 30 μmol/L of GS can lead to TEMT in HK-2 cell.The QLT0267 with concentration greater than 5 μmol/L may prevent the activation of ILK downstream proteins,then partially inhibits cell proliferation and TEMT in HK-2 cell.

Objective To investigate the effects of 1,25-dihydroxyvitamin D3[1,25 (OH)2D3] on cell proliferation in hu?man glomerular mesangial cells and it′s effects on the regulation of mTOR/p70s6K signaling pathway in this cell line. Meth?ods The cultured human mesangial cells at passage 3-7 were divided into four groups:control group,VD group (addition of 10-8 mol/L of 1,25-dihydroxyvitamin D3 ),R group (addition of 5 mg/L of rapamycin) and R+VD group(addition of 5 mg/L ra?pamycin combined with 10-8 mol/L of 1,25-dihydroxyvitamin D3). Drug incubation last 48 h. The effect of mesangial cell pro?liferation was measured by CCK-8 colorimetric assay. The cell cycles were measured by flow cytometry. The expression of mTOR and p70s6K were detected by immunofluorescence. Results (1) The absorbance of A450 was higher in control group than that in VD group than that in R group than that in R+VD group. But the inhibition rate (IR) was lower in control group than that in VD group than that in R group than that in R+VD group. All comparisons were of statistic significance. ( 2) Cells in G1 phase were higher while cells in G2/M and S phases as well as proliferation rate (PI) were lower in control group than those in VD group than those in R group than those in R+VD group. All comparisons were of statistic significance except in?dexes between group R and group VD. (3) mTOR and p30s6K expressions in mesangial cells were higher in control group than those in VD group than those in R group than those in R+VD group. All comparisons were of statistic significance ex?cept indexes between group R and group VD. Conclusion 1,25-dihydroxyvitamin D3 might inhibit mesangial cell prolifera?tion significantly through mTOR/p70s6K signaling pathways.

Purpose　Both the working standard and corresponding sample(GRAN75)were first calibrated by WHO international standard for G-CSF.Methods　MTT method by NFS-60 cells was used. The results were calculated by (4,4)method.Results　Three batchs working standards were prepared,two batchs were freeze-dry and the prescription was same as WHO international standard for G-CSF,one batch was injection and the prescription was same as corresponding sample(GRAN75).The biological potency and the FL% of average potency were 3.062×106 、4.276×106IU/ampoule、1.635×107IU/ml and 5.529%、4.291%、4.244% for working standard, and 1.880×107IU/ml and 5.175% for corresponding sample,respectively.Conclusion　The working standard which calibrated could be used as working standard in the measurement of rhG-CSF biological activity.

Purpose　The determination method of rhG-CSF bio logical activity by NFS-60 cells was studied in accordance with WHO internation al standard for G-CSF.Methods　MTT method was adopted.R esults　The chomosone number of NFS-60 cell was 39, about 1×105 cells/ml to 1×107 cells/ml,the dying color showed gradient when the NFS-60 c ells was dyed by MTT,the method(4,4) was adopted in the determination of rhG-CS F biological activity,the average FL% of potency was 13.560% for single exprimen t,and the CV of inter-assay and intra-assay was lower than 10% and 10.109%,respectively.Conclusion　method(4,4) can be us ed in the determination of rhG-CSF biological activity,and the results can guid e the study and the manufacture of rhG-CSF effectively.