Purpose To construct a lentiviral ventor-mediated RNA interference of human midkine ( MK) gene and to provide the ba-sis for further experiment in vivo and in vitro. Methods Four RNAi sequences targeting human MK gene were designed, and cloned into the lentiviral vector to construct lentiviral vectors:GV115-MK-1, GV115-MK-2, GV115-MK-3 and GV115-MK-4. After transfec-tion into competent E. Coli bacteria, the candidate clones were identified by PCR and DNA sequencing. The titer of lentiviruses was determined after 293 T cells were co-transfected with GV115-MK, pHelper 1. 0 and pHelper 2. 0. The four kinds of recombinant lenti-viruses were used to infect human breast cancer cell MDA-MB-231, and the expression levels of MK mRNA were detected by real-time PCR. Results PCR analysis and DNA sequencing confirmed that the four inserted MK RNAi sequences were corrected. Strong green fluorescence was observed in the 293 T cells under the fluorescent microscope after co-transfection. The titer of the concentrated virus was 6 × 108 , 5 × 108 , 5 × 108 and 6 × 108 TU/ml, respectively. The MK expression in MDA-MB-231 cells infected with lentiviral vec-tors GV115-MK-1 was decreased by 87. 2% (P<0. 05). Conclusion Lentiviral RNAi vector for MK gene is successfully construc-ted, and it could effectively inhibit the expression of MK gene in MDA-MB-231 cells in vitro.

Since the early symptoms are very hidden,most of patients with colorectal cancer (CRC)have occurred metastasis when diagnosed.Recently,chemotherapy combined with targeted therapy is the major therapeutic strategy for metastatic CRC and the high frequency activation of phosphatidylinositol 3-kinase (PI3K) signaling pathway has been validated sharing a close relationship with the development of CRC,contributing important values for the diagnosis and therapy of CRC.

ObjectiveTo investigate the intervention of level of atorvastatin calcium tablets (lipitor) on soluble CD40 (CD40L) and high sensitivity C-reactive protein (hs-CRP) in patients with angina.Methods 65 patients with coronary heart disease were divided into stable angina (SA group,n= 30) and unstable angina (UA group,n= 35).The vein blood was collected in the hospitalized morning,2,4,6 weeks after treatment and serum levels of hs-CRP and CD40L were measured and compared between the two groups.ResultsThe levels of soluable CD40L [(20.52± 2.91)μg/L] and CRP [(7.96±1.69) mg/L] in UA group were higher than SA group [(7.96±-1.35) tg/L and ( 1.58 ± 0.91 ) mg/L] (t = 21.705、18.493,both P＜ 0.05) before treatment,and after treatment,their levels in the two groups were significantly lower than pre-treatment (P＜0.01).ConclusionsCD40L and hs-CRP may involved in the pathophysiology of unstable angina process and can be used as an indicator reflecting vulnerable plaque.Lipitor might enhance stability of atherosclerotic plaque and prevent acute coronary events by reducing levels of CD40L and hs-CRP.

Purpose To observe the effect of MK on proliferation, migration and angiogenesis of human umbilical vein endothelial HU-VECs cells and to explore the role of MK in tumor angiogenesis. Methods siRNA targeting MK was used to downregulate MK expres-sion in human breast cancer cell line MDA-MB-231. Then, the experiment was divided into three groups: the untreated group, the empty vector transfection group and MK gene interference group. CCK-8 assay was used to detect the endothelial cells proliferation, tr-answell method was for detection of endothelial cell migration numbers, and matrigel in vitro small tube formation assay was used to sur-vey the state of tube formation. Expression of MK in siRNA transfection was identified by RT-PCR and Western blot. Results The MK gene interference group showed lower cell proliferation activity, less number of migration of cells and tube formation than the other two groups (P<0. 05). Conclusion Low-expressed MK in human breast cancer cell line MDA-MB-231 can inhibit proliferation, mi-gration and angiogenesis of endothelial cells, which shows that MK may play an important role in tumor angiogenesis.

Objective To investigate the adjustment and value of lymphocyte G protein-coupled receptor kinase-2 (GRK2) protein levels in patients with acute coronary syndrome(ACS). Methods Forty-two patients with stable angina pectoris (SAP), 44 patients with unstable angina pectoris (UAP), 43 patients with acute myocardial infarction (AMI), and 46 patients with normal coronary angiography (NCA) in hospital were enrolled in this study. Lymphocyte GRK2 protein levels were analyzed by Western blot in 24 h after admitted to hospital. Heart rate variability (HRV) analysis was performed based on 24 h Holter electrocardiogram (ECG) monitoring. Cardiac functions were measured using ultrasonic cardiogram. The results were compared and the relationship between GRK2 protein levels and HRV, cardiac functions index was analyzed. Results The level of lymphocyte GRK2 in AMI group, UAP group, SAP group and NCA group was (209.8 ± 63.9)%, (165.6 ± 60.2)%, (131.7 ± 51.8)% and (125.3 ± 50.6)%. The levels of lymphocyte GRK2 in AMI group and UAP group was significantly higher than that in SAP group and NCA group .Moreover, the level of GRK2 was the highest in AMI group, and there were significant differences (P<0.01). The level of lymphocyte GRK2 had negative correlation with high frequency(HF), low frequency(LF), LF/HF, standard deviation NN interval (SDNN), standard deviation of the average normal RR interval for 5-minute segments (SDNNI) and left ventricular ejection fraction (LVEF) (r =-0.52,-0.47,-0.53,-0.56,-0.49,-0.51, P < 0.01). Conclusions The rise of lymphocyte GRK2 protein levels is significantly associated with increased sympathetic nerve excitability and deterioration of cardiac function.

Objectives To investigate the effects of sodium arsenite (NaAsO2) on the expression ot microRNA-191 (miR-191) and tissue inhibitor of metalloproteinase 3 (TIMP-3) in human normal hepatic cells (L-02 cells).Methods L-02 cells were exposed to different doses of NaAsO2 [0 (control group),5,25,50 and 75 μmol/L]for 24 h,or treated with 5 and 25 μmol/L NaAs02 for 0 (control group),12,24 and 48 h.The miR-191 inhibitor was used to suppress the expression of miR-191.qRT-PCR was performed to detect the expression level of miR-191 and TIMP-3 mRNA,and the protein level of TIMP-3 was analyzed by Western blotting.Results Dose-effect study:There were significant differences in the expressions of miR-191,TIMP-3 mRNA and protein between the 5 groups (F =85.674,20.952,123.393,all P ＜ 0.05).The expressions of miR-191 in all groups (1.702 ± 0.124,2.077 ±0.234,2.145 ± 0.105,2.003 ± 0.077) were higher than that of control group (0.990 ± 0.035,all P ＜ 0.05);the mRNA expressions of TIMP-3 in 25,50,75 μmol/L groups (0.848 ± 0.067,0.804 ± 0.081,0.813 ± 0.076) were all lower than that of control group (0.996 ± 0.007,all P ＜ 0.05),but there was no significant difference in the mRNA expression of TIMP-3 between the 5 μmol/L group and control group (0.939 ± 0.133 vs 0.996 ± 0.007,P＞ 0.05),and the protein expressions of TIMP-3 in all groups (0.846 ± 0.093,0.611 ± 0.123,0.554 ± 0.098,0.529 ± 0.067) were lower than that of control group (1.006 ± 0.003,all P ＜ 0.05).Time-effect study:there were significant differences in the expressions of miR-191,TIMP-3 mRNA and protein between the exposure groups of 5 and 25 μmol/L (For 5 μmol/L:F =86.355,16.404,22.898,all P ＜ 0.05;For 25 μmol/L:F =104.321,20.123,52.321,all P ＜ 0.05).The expressions of miR-191 in all exposure groups of 5 and 25 μmol/L (1.392 ± 0.152,1.691 ± 0.167,2.018 ± 0.130 and 1.456 ± 0.167,1.946 ± 0.178,2.259 ± 0.256) were higher than those of control groups (1.001 ± 0.014,1.008 ±0.027,all P ＜ 0.05);the mRNA expressions of TIMP-3 in 48 h exposure group of 5 μmol/L and all exposure groups of 25 μmol/L (0.824 ± 0.093 and 0.897 ± 0.033,0.815 ± 0.089,0.709 ± 0.103) were lower than those of control groups (1.004 ± 0.018,0.997 ± 0.057,all P ＜ 0.05),but there were no significant differences in the mRNA expressions of TIMP-3 between the 12,24 h exposure groups of 5 μmol/L and control group (0.952 ± 0.072,0.929 ± 0.121 vs1.004 ± 0.018,all P ＞ 0.05);the protein expressions of TIMP-3 in all exposure groups of 5 and 25 μmol/L (0.857 ±0.068,0.832 ± 0.106,0.691 ± 0.112 and 0.785 ± 0.097,0.620 ± 0.066,0.453 ± 0.075) were lower than those of control groups (1.006 ± 0.045,1.004 ± 0.078,all P ＜ 0.05).The treatment of miR-191 inhibitor:there were significant differences in the expressions of miR-191 and TIMP-3 protein between different groups (F =104.306,67.015,all P ＜ 0.05).The elevated expression level of miR-191 induced by NaAsO2 was significantly suppressed after transfected with miR-191 inhibitor (0.314 ± 0.094 vs 2.051 ± 0.371,P ＜ 0.05),which in turn up-regulated the protein expression of TIMP-3 (1.965 ± 0.277 vs 0.541 ± 0.183,P ＜ 0.05).Conclusion The expression level of miR-191 is elevated in response to NaAsO2 exposure,and miR-191 has subsequently suppressed the expression of TIMP-3,a potential target of miR-191.

Objective To investigate the feasibility and clinical effect for percutaneous microwave ablation of type I substernal goiter under the guidance of ultrasound . Methods Thirteen patients with type I substernal goiter were selected . All patients underwent percutaneous microwave ablation treatment under the guidance of ultrasound . Thirteen patients with 47 medals nodules were performed ultrasound guided percutaneous microwave ablation . Intraoperative heat blocking blood flow was used to prevent severe hemorrhage .Liquid isolation belt and leverage from methods were used to effectively prevent surrounding important structures against heat damage .Small amount of residual method was used to avoid tracheal collapse . All patients were followed up post‐operative 1 ,3 ,6 ,12 months by monitoring of thyroid nodule volume change , adverse reactions , and postoperative complications . Results Thirteen patients with 47 medals nodules were successfully performed percutaneous microwave ablation guided by ultrasound . Among them ,2 patients performed the second treatment for too large volume of goiter ,tracheal displacement and poor physical tolerance of older . The postoperative ultrasound contrast and color Doppler showed that the lesions were completely inactivated . No obvious complications occurred in all 13 patients , and no permanent hoarseness occured . The symptoms of cervical oppression and discomfort disappeared for all patients within 1-4 months after surgery . Thirteen patients were followed up for 12 months after the operation . The thyroid function was normal and the volume reduction rate of thyroid nodules was (85 ± 31)% . Conclusions Percutaneous microwave ablation of type I substernal goiter under ultrasonic guidance is a safe and effective method to reduce the thyroid nodules with no serious complications . It is worth to be popularized in clinical practice .

Aim Through interfering the expression of survivin with short hairpin RNA(shRNA) technology,to investigate the effect of downregulation of survivin on cell apoptosis and chemosensitivity to docetaxel in breast cancer MCF-7 cells.Methods(1) Using MCF-7 cells as a model system,three groups were set up transfected with lipofectamine,RNAi control plasmid and survivin RNAi plasmid,respectively.The expression of survivin in MCF-7 cells was measured at transcriptional and translational level by using RT-PCR and Western blot methods.(2) The effect on the cell cycle and apoptosis was analyzed with flow cytometry.(3) The viability of cells applied with different doses of ducetaxel was determined by using the method of 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction(MTT method).Results(1) RT-PCR and Western blot demonstrated that survivin expression was significantly decreased by transfection with RNAi targeting plasmid;the expression proportion was reduced by nearly 75.4% and 79.8%(P

Objective Through comparing ultrasound-guided microwave ablation (MWA) with surgical resection for the treatment of nodular goiter to evaluate the effect of RFA on the thyroid function.Methods A total of 50 patients with nodular goiter,who were admitted to authors' hospital during the period from January 2010 to January 2013 to receive ultrasound-guided MWA,were selected and used as the study group;and other 96 patients with nodular goiter,who were encountered at authors' hospital during the same period to receive partial thyroidectomy,were collected and used as the control group.Of the 50 patients in the study group,RFA of unilateral lobe nodules was performed in 8 and RFA of bilateral lobe nodules in 42.Among the 96 patients in the control group,unilateral lobe nodules were seen in 52 and bilateral lobe nodules in 44.Postoperative complications were recorded,the serum FT3,FT4,TSH levels were determined at one week,as well as one,3,6 and 12 months after the treatment.The results were statistically analyzed.Results All patients were followed up for 12 months.In the study group,no complications occurred;one week after RFA the serum FT3 and FT4 levels increased while the serum TSH level decreased when compared with preoperative ones,and the differences were statistically significant (P＜0.05).In the control group,bleeding asphyxia occurred in one patient,hoarseness in 5 patients and hypocalcemia convulsion in 3 patients.One week after partial thyroidectomy,the serum FT3 and FT4 levels increased while the serum TSH level decreased when compared with preoperative data,and the differences were statistically significant (P＜0.05).One week after surgery thyroxine replacement therapy was routinely given to 44 patients who had received bilateral subtotal thyroidectomy.Three patients who had received unilateral subtotal thyroidectomy developed hypothyroidism at 3,6 and 8 months after the surgery respectively.In other 49 patients receiving unilateral subtotal thyroidectomy the serum FT3,FT4,TSH levels determined at one,3,6 and 12 months after surgery were significantly different from those in the patients of the study group (P＜0.05).Conclusion For the treatment of nodular goiter,ultrasound-guided MWA is quite safe.MWA has very slight effect on thyroid function and is definitely superior to subtotal thyroidectomy.Therefore,MWA is a minimally invasive technique which is worthy of clinical promotion.

Purpose To observe the effects of midkine ( MK) on human breast cancer cell line MDA-MB-231 angiogenesis in vitro, and to explore its mechanism. Method shRNA interference was performed to silence the expression of MK in MDA-MB-231 cells, and Western blot was used to identify the expression of MK and EPCR. After MK and EPCR knockdown, or treated with anti protease-activated receptor 1 (PAR1) antibody, the culture medium of MDA-MB-231 cells were collected and the conditioned medium were pre-pared. Human umbilical vein endothelial cells ( HUVECs) were cultured with conditioned medium, and the endothelial cells prolifera-tion was detected by CCK-8 assay, cell migration was detected by transwell method, vasculogenic activity was assessed by Matrigel-based tube formation assay. Results After knockdown of MK, the protein level of EPCR was decreased in MDA-MB-231 cells. Com-pared with control, knockdown of MK and EPCR decreased the proliferation, migration and angiogenesis ability of HUVECs significant-ly (P<0. 05), and the effect of EPCR knockdown group was stronger than MK knockdown group (P<0. 05). After treated with anti-PAR1 antibody, the proliferation, migration and angiogenesis ability of HUVECs were decreased compared with control and EPCR knockdown group (P<0. 05). Conclusion MK promotes human breast cancer cell line MDA-MB-231 angiogenesis through EPCR /PAR1 signaling pathway in vitro.