10.3969/j.issn.2095-4344.2013.24.020

Objective To construct a highly efficient expression plasmid of eukaryotic nuclear membrane protein Omega 3 fatty acid desaturase gene Fat‐1 in E .coli .Methods Using molecular cloning technology to construct the recombinant prokaryotic expression plasmid pET32a Fat‐1 and pET32a‐Mistic‐Fat‐1 fused with Membrane proteins expression chaperon mistic ;the two re‐combinant plasmids were transformed into E .coli strain BL21 (DE3) ,the expression of Fat‐1 protein and M110 Fat‐1 protein in‐duced by IPTG were identified by SDS‐PAGE and gray degree analysed the amount of expression ,further identified by Western blot .Results The results of enzyme digestion and sequencing demonstrated that we successfully constructed the prokaryotic ex‐pression vectors pET32a Fat‐1 and pET32a‐Mistic‐Fat‐1;SDS‐PAGE and Western blot showed that Fat‐1 fatty acid desaturase wasn′t significantly induced ,but the overexpression of M110 Fat‐1 fusion protein was obtained in E .coli ,accounting for 15% of the total amount of whole cell proteins .Conclusion The fusion with Mistic proteins to express the Fat‐1 gene has realized the overex‐pression of eukaryotic nuclear membrane integrated protein Omega 3 fatty acid desaturase in prokaryotic host .

Objective To improve the diagnosis and treatment of asymptomatic adrenal pheochromocytoma. Method The clinical data of 33 patients with asymptomatic adrenal pheochromocytoma were reviewed. Results All the patients were not preut with typical signs and symptoms of pheochromocytoma, 16 patients were found adrenal mass by routine physical examination, 15 patients presented with superior abdominal or flank pain, 1 patient was fever and 1 patient was diarrhea. Most of patients were prent with round mass with low density in the center of the tumor,5 patients had elevated level of plasma epinephrine and norepinephrine (16% ,5/31),24 hours urine CA and VMA were elevated in 6 patients (24%, 6/25) and 5 patients (20%, 5/25) respectively, 26 patients who were suspected pheochromocytoma preoperatively were treated with α-adrenergic blockade (prazosin) to control the blood pressure or with intravenous colloid fluids and blood transfusion to expand intravascular volume before operation. The tumors were removed successfully in 32 patients, and biopsy was done in 1 patient because of the serious invasion to the vena cava by the tumor. All the patients were diagnosed pheochromocytoma histopathologically,and followed up for 1 month to 7 years, no tumor recurrence. Conclusions The patients with asymptomatic adrenal pheochromocytoma have lower catecholamine excretion in their plasma and urine. Combination of modem imaging examinations are useful methods to diagnose asymptomatic adrenal pheochromocytoma. To lower the risk of operation, the control of blood pressure and the expanding of intravascular volume are important for the patients with huge adrenal mass or typical suspected appearance of pheochromocytoma.

Objective The aim of this study was to generate human cholesteryl ester transfer protein ( CETP) transgenic rabbits and analyze their biological properties.Methods We generated human CETP transgenic rabbits by DNA microinjection, and detected the expression of human CETP by real-time PCR and Western blot assay.The activity of CETP was measured using an activity assay kit.Results Human CETP transgenic rabbits were successfully generated by DNA microinjection.Compared with wide type rabbits, the expression of human CETP was dramatically increased in the liver of the human CETP transgenic rabbits.The plasma CETP activity was also much higher in the liver of human CETP transgenic rabbits than that of control rabbits.Conclusions The model of human CETP transgenic rabbits is successfully established by DNA microinjection.It will provide a useful tool for the studies of CETP biological function and its involvement in the mechanisms of cardiovascular diseases.

Objective To investigate the regulation of T cells in the process of liver regeneration using a model of mice after 70% liver resection.Methods We performed 70% hepatectomy in T-cell-deficient mice and control mice.The liver mass and body mass ratio, BrdU infiltration level, proliferating cell nuclear antigen (PCNA),expression of M phase marker protein p-HDAC3, and serum transaminase levels were measured.Results The recovery of liver mass and body mass ratio of thymus-deficient mice occurred significantly later than that of control mice.The peak time of BrdU infiltration levels and the expression of PCNA and p-HDAC3 in T-cell-deficient mice were later than in control mice, but the degree of liver injury was lower.Conclusion T cells are involved in the regulation of liver regeneration, and the absence of T cells delays the process of liver regeneration.

AIM:To investigate the effect of quercetin on the biological functions of rat bone marrow-derived endothelial progenitor cells (EPCs) and its potential mechanisms.METHODS:The bone marrow-derived mononuclear cells of Sprague-Dawley rats were isolated by density gradient centrifugation.The differentiated EPCs were cultured specially and stained with DiI-Ac-LDL and FITC-UEA-1.CD133+ and FLK-1+ were detected on the cell surfaces.After 14 d, the EPCs were incubated with a PI3K inhibitor BYL719 (3 μmol/L) and an ERK inhibitor FR180204 (15 μmol/L).After incubation of the inhibitors for 2 h, the cells were treated with quercetin at different concentrations (0, 10, 20, 40, 80 and 100 μmol/L).MTT assay and Transwell assay were used to detect cell viability and the number of migratory cells.The protein levels of AKT, eNOS, ERK and their phosphorylated status were determined by Western blot.RESULTS:Quercetin enhanced the viability and migration of the EPCs at a dose-dependent manner.However, the PI3K inhibitor BYL719 suppressed the QUE-induced cell viability and migration.Moreover, ERK inhibitor FR180204 exerted the similar inhibitory effect on the cell viability but had no effect on cell migration.Quercetin activated the phosphorylation of AKT, eNOS and ERK.On the other hand, BYL719 was observed to inhibit the phosphorylation of AKT and ERK.FR180204, however, was showed to inhibit the phosphorylation of ERK only.On the contrast, the stimulatory effects that quercetin exerted on the expression of eNOS and its phosphorylation were suppressed by BYL719 and FR180204.CONCLUSION:Quercetin stimulates the viability and migration of EPCs via PI3K/AKT/eNOS and ERK/eNOS signaling pathway, which would be beneficial for cardiovascular health.

Objective To evaluate the left ventricle systolic function in patients with aortic stenosis (AS) underwent transcatheter aortic valve implantation(TAVI) by speckle tracking imaging and to observe the indicators in bicuspid aortic valves(BAV) and tricuspid aortic valves(TAV) groups.Methods Twenty nine patients with AS were enrolled,all of them underwent TAVI successfully.The regular echocardiography and 3D full-volume images were acquired on before and 3 days,1 month after TAVI.Longitudinal strain,circumferential strain,and three-dimensional left ventricle ejection fraction(3D-LVEF) were analyzed using Qlab software.Results Compared with the baseline,aortic valve blood flow velocity (AV),maximum aortic valve pressure gradient (AVPG-max),mean aortic valve pressure gradient (AVPG-mean),aortic valve area(AVA) after TAVI were improved significantly.Global longitudinal strain(GLS) had a improvement on 3 days after TAVI(all P ＜0.001),and further increased during 1 month after TAVI (all P ＜0.001).Global circumferential strain(GCS) were increased during 1 month after TAVI(all P ＜ 0.001).The 3D-LVEF after 1 month were improved significantly(all P ＜0.001).The BAV patients and TAV patients had similar changes in all of indicators observed.Conclusions The left ventricle systolic function has early improvement after TAVI,and further recovery during follow-up.The BAV patients can obtain a benefit from TAVI equally to the TAV patients.

Objective To investigate the expressions of Wnt3a and Wnt5a in papillary thyroid carcinoma (PTC) and their clinical significances. Methods Immunohistochemical SABC method was used to detect the expressions of Wnt3a and Wnt5a proteins in PTC tissues and their paracancerous tissues collected from 79 patients in Dandong First Hospital from January 2014 to June 2018, and the relationships between the expressions of Wnt3a and Wnt5a proteins and clinicopathological features of PTC patients were analyzed. The expressions of Wnt3a and Wnt5a proteins in 10 pairs of fresh PTC tissues and paracancerous tissues were detected by Western blot. Results The results of immunohistochemistry showed that the positive expression rates of Wnt3a and Wnt5a proteins in PTC tissues were significantly higher than those in paracancerous tissues [69.6％ (55/79) vs. 25.3％ (20/79), 60.8％ (48/79) vs. 20.2％ (16/79)], and the differences were statistically significant (χ 2 values were 31.092 and 26.894, both P < 0.01). The results of Western blot showed that the expressions of Wnt3a and Wnt5a proteins in 10 pairs of fresh PTC tissues was significantly higher than those in paracancerous tissues(1.61±0.40 vs. 0.43±0.14, 1.38±0.291 vs. 0.36±0.13), and the differences were statistically significant (t values were 16.234 and 13.493, both P < 0.01). The expressions of Wnt3a and Wnt5a in PTC tissues were correlated with TNM stage, differentiation, extramembranous invasion and lymph node metastasis (Wnt3a: χ2 values were 6.645, 15.945, 8.783 and 11.220; Wnt5a: χ2 values were 21.525, 7.611, 17.880 and 12.581, all P < 0.05), but not with patients'age, sex and tumor diameter (all P > 0.05). There was a positive correlation between Wnt3a and Wnt5a proteins expressions in PTC (r = 0.597, P < 0.01).Conclusion The abnormal expressions of Wnt3a and Wnt5a proteins in PTC may be related to the development of PTC.

Objective: To investigate the expression of HOXA terminal transcript antisense RNA (HOTTIP) in hepatocellular carcinoma (HCC) tissues and to explore its effect on proliferation, invasion and migration in HepG2 cells. Methods: A total of 60 cases with HCC tissues undergoing excision surgery and 60 cases of corresponding paracancerous tissues from January 2012 to June 2018 in Dandong First Hospital of Liaoning Province were collected. The expressions of HOTTIP in HCC tissues and paracancerous tissues were detected by using real-time quantitative polymerase chain reaction (RT-qPCR), and the relationship between the expression and clinicopathological features was analyzed. HepG2 cell line with high expression of HOTTIP constructed by cell transfer technique was treated as the experimental group, and the empty plasmid pcDNA3.1-NC was treated as the control group. The effect of HOTTIP on the proliferation of HepG2 cells was detected by using CCK-8 method, and the effect of HOTTIP on invasion and migration of HepG2 cells was detected by using Transwell assay. Results: The expression of HOTTIP mRNA in HCC tissues was higher than that in paracancerous tissues, and there was no statistically significant difference (1.9±0.6 vs. 0.9±0.7, t = 6.069, P < 0.01). The whole HCC cases were divided into the high expression group (30 cases) and the low expression group (30 cases) according to the median value (1.92) of the expression of HOTTIP mRNA. The expression of HOTTIP was related with TNM stage, differentiation degree and lymph node metastasis (χ² values were 10.800, 8.076, 5.711, all P < 0.05), but not with age, gender, tumor diameter, number of tumors, hepatitis and alpha fetoprotein (AFP) levels (all P > 0.05). The results of RT-qPCR showed that the expression of HOTTIP mRNA in HepG2 cells was increased after transfection of overexpressed HOTTIP and the differences was statistically significant compared with the control group (63±6 vs. 13±9, t = 9.129, P < 0.01). The results of CCK-8 method showed that the proliferation activity of cells was enhanced after the overexpression of HOTTIP in HepG2 cells (24 h, 36 h, 72 h at 490 nm absorbance was 1.497 ± 0.017 vs. 0.826±0.006, 2.002±0.025 vs. 1.211±0.020, 3.257±0.042 vs. 1.772±0.021), and the differences were statistically significant (t values were 5.321, 7.349, 8.793, all P < 0.01). In Transwell invasion assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (101±9 vs. 41±11), and the difference was statistically significant (t = 6.839, P < 0.01). In Transwell migration assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (112±9 vs. 53±11), and the difference was statistically significant (t = 7.105, P < 0.01). Conclusion The expression of HOTTIP in HCC tissues is up-regulated, and the overexpression of HOTTIP can promote the proliferation, invasion and migration of HCC cells.

Objective: To investigate the effects of transcatheter aortic valve replacement (TAVR) in patients with severe aortic valve stenosis. Methods: The clinical data of 130 patients with severe aortic valve stenosis, who underwent TAVR in our hospital with self-expanding valve between January 1,2010 and October 30, 2016, were analyzed retrospectively. The patients were divided into calcific aortic valve stenosis (CAS) group (112 cases) and non-calcific aortic valve stenosis (NCAS) group (18 cases) according to 3D volume-rendering reconstruction under multiple detector computed tomography before TAVR. The baseline clinical features, imageology results, procedural details, and clinical prognosis were compared between the 2 groups. Results: (1) Compared with CAS group, the patients in NCAS group were younger, had higher proportion of rheumatic heart disease, and less proportion of bicuspid aortic valve morphology (P<0.01 or 0.05). Except for minimum value of sinotubular junction (P=0.017), there were no significant differences in multiple detector computed tomography measurements of aortic valve annulus and aortic root structure between the 2 groups (all P>0.05). (2) Compared with CAS group, the proportions of valve release under rapid pacing and oversized valve release were larger than pre-procedural evaluation, and the proportion of post-dilation was lower in NCAS group (P<0.01 or 0.05). (3) Post-procedural transthoracic echocardiography revealed that left ventricular ejection fraction was higher than baseline level in CAS group (P<0.001), while which was similar in NCAS group (P=0.552). Compared with before TAVR, mean pressure gradient and maximum transvalvular velocity were significantly reduced, aortic valve orifice area was significantly increased, and proportion of moderate to severe aortic regurgitation was significantly reduced after the procedure in both groups (all P<0.01). There were no significant differences in left ventricular ejection fraction, mean pressure gradient, maximum transvalvular velocity, aortic valve orifice area, and proportion of moderate to severe aortic regurgitation after TAVR between the 2 groups (all P>0.05). (4) There were no significant differences in successful rate of device placement and cardiovascular related death within 30 days after TAVR between the 2 groups (105/112 vs. 17/18, P=0.909; 3/112 vs. 1/18, P=0.453, respectively). Conclusion TAVR is safe and effective for patients with severe aortic valve stenosis.