Biobanking of fresh-frozen human colon tissues: impact of tissue ex-vivo ischemia times and storage periods on RNA quality.

BACKGROUND: Biobanking plays an important role in translational cancer research. The impact of tissue ex-vivo ischemia time and storage period on RNA integrity is not well documented. METHODS: Fresh-frozen colon tissues were collected in Taizhou Hospital of Zhejiang Province in China since 2004. Fifty-one colon cancer tissues with tumor cell content higher than 70 % and matched normal tissues during four storage periods (less than 15 months, 16-20 months, 21-25 months, and 26-40 months) were chosen to detect RNA quality. Fresh colon cancer tissues from 5 patients were cut into pieces and kept at room temperature or on ice for 0.5, 1, 2, and 4 h before snap freezing. RNA integrity was determined by microcapillary electrophoresis by the RNA integrity number (RIN) algorithm. RESULTS: Sixty-seven percent of normal colon tissues and 94 % of colon cancer specimens yielded RNA with a RIN of ≥7. Matched colon cancer and normal tissues showed significant difference in RNA quality. RNA remained stable in colon cancer tissues kept at room temperature and on ice for up to 4 h, and long-term storage of banked colon specimens did not negatively influence RNA quality (RNA with RIN of ≥7 banked less than 15 months, 83 %; 16-20 months, 78 %; 21-25 months, 77 %; 26-40 months, 90 %). CONCLUSIONS: Frozen colon tissues yield high-quality RNA in approximately 80 % of specimens. Ex-vivo ischemia times and storage periods did not adversely affect RNA quality. This study showed that standard operation protocols and the maintenance of high-quality tissue repositories were the keys to translational medicine research.

Plasma soluble human leukocyte antigen-G expression is a potential clinical biomarker in patients with hepatitis B virus infection.

The significance of upregulated soluble human leukocyte antigen-G (sHLA-G) expression under various pathologic conditions has been discussed. In this study, we evaluated the potential significance of plasma sHLA-G expression in patients with hepatitis B virus (HBV) infection. The study included 90 acute hepatitis B patients (AHB), 131 chronic hepatitis B patients (CHB), 152 resolved hepatitis B individuals (RHB), and 129 normal controls. sHLA-G were determined using enzyme-linked immunosorbent assay. A receiver operating characteristic (ROC) curve was used to evaluate the feasibility of plasma sHLA-G as a biomarker for distinguishing patients with HBV infection. sHLA-G levels in AHB (median, 193.1 U/mL; p < 0.001), CHB (median, 324.6 U/mL; p < 0.001), and RHB (median, 14.8 U/mL; p = 0.006) patients was much higher than that in normal controls (median, 9.0 U/mL). A significant difference for sHLA-G levels was also observed between patients with HBV infection (AHB vs CHB, AHB vs RHB, and CHB vs RHB; all p < 0.001). The area under the ROC curve for sHLA-G levels was 1.000 (p < 0.001) for AHB, 0.993 (p < 0.001) for CHB, and 0.604 (p = 0.003) for RHB patients versus normal controls, respectively. Data also indicated that the percentage of CD4(+)CD25(+)FoxP3(+) T regulatory cells and HLA-G(+)CD14(+) monocytes was significantly increased in AHB and CHB patients compared with normal controls (all p < 0.001). Our findings indicated that induction of HLA-G expression may play a role in HBV immune evasion and sHLA-G levels could be a useful biomarker in HBV infection.

[Angiogenesis in chronic ischemic porcine myocardium after transfer of VEGF165 and angiopoietin-1 mediated by recombinant adeno-associated viral vector].

OBJECTIVE: To study the effects of combination of angiopoietin-1 (ANG-1) and vascular endothelial growth factor165 (VEGF165) gene transfer mediated by recombinant adeno-associated viral vector on the neovascularization in chronic ischemic porcine myocardium. METHODS: An ameroid constrictor was implanted around the left circumflex coronary artery (LCX) via endoscopy. Six weeks later, coronary angiography revealed that the myocardial ischemia was established by gradual occlusion of the left circumflex coronary artery (LCX). Sixteen swine with the total occlusion or partial stenosis (> 85 %) of the LCX were divided into 4 groups (4 in each group): group I, group II and group IV (control) received direct myocardium injection of rAAV2 VEGF165, rAAV2 ANG-1 or PBS alone, respectively; group III received rAAV2 VEGF165 and rAAV2 ANG-1. Selective coronary angiography and ultrasonography were performed perioperatively to evaluate the cardiac function and the formation of collateral circulation. The expression of VEGF165 and ANG-1 proteins were assessed using ELISA or Western blot. The degree of angiogenesis was assessed by use of immunohistochemical analysis. RESULT: Angiography showed that the occlusion of all LCX was completed or exceeded 95% 6 weeks after ameroid constrictor implantation, indicating the successful establishment of animal model. The expression levels of VEGF165 in group I and III and ANG-1 in groups II and III began to increase at d7 after transfection and reached the peak at d14; then decreased gradually to the normal level after 3 months. The expression levels of VEGF165 in group II and group IV or that of ANG-1 protein in group I and group IV had no markedly changes at different time after transfection. There were significant increase in capillary density and arteriole density and more side branch vessels formed in group III compared with other groups. Echocardiographic measurements showed that the left ventricular systolic function of animals in groups I, II and III increased significantly after gene transfection, especially in group III; but there was no changes in group IV. CONCLUSION: Myocardial perfusion and the left ventricular systolic function are improved after rAAV2 VEGF165 or rAAV2 ANG-1 transfection, which is associated with the angiogenesis in porcine model of chronic myocardial ischemia.

Analysis of the plasma soluble human leukocyte antigen-G and interleukin-10 levels in childhood atopic asthma.

Human leukocyte antigen-G (HLA-G) has been hypothesized to be associated with the pathogenesis of asthma; however, results remain controversial. Furthermore, HLA-G expression could be modulated by the HLA-G 14-bp insertion (+)/deletion (-) polymorphism and by interleukin-10. In this study, the 14-bp polymorphism in exon 8 of the HLA-G gene, plasma soluble HLA-G, and interleukin-10 (IL-10) levels in untreated atopic asthmatic children, and in a group of age-, gender-, and ethnicity-matched normal controls were analyzed. Data showed that HLA-G 14-bp +/- polymorphism was not significant difference between the asthmatic patients and normal controls. Plasma soluble human leukocyte antigen (sHLA)-G in atopic asthma patients (n = 72; median, 179.28 U/ml) was dramatically higher compared with that of the normal controls (n = 76; median, 35.23 U/ml; p < 0.001). Receiver operating characteristic (ROC) curve analysis showed that the area under ROC curve for sHLA-G was 0.986 (p < 0.001) in atopic asthma patients versus normal controls. IL-10 levels in the asthmatic children (n = 50; median, 5.02 pg/ml) was significantly lower than that of the normal controls (n = 48; median, 12.82 pg/ml; p < 0.001). Both HLA-G 14-bp polymorphism and IL-10 levels were unrelated to plasma sHLA-G concentration in both groups. Our findings indicated that the HLA-G 14-bp polymorphism was not a risk factor, but that sHLA-G might be considered as a biomarker for the atopic asthmatic patients. Dramatically increased sHLA-G with decreased IL-10 levels may have implications in the pathogenesis of atopic asthma.

[Construction of adeno-associated virus vector containing ANG-1 gene and its expression in pig mesenchymal stem cells].

OBJECTIVE: To construct recombinant adeno-associated virus (rAAV) vector containing angiopoietin-1 (ANG-1) gene and to express the ANG-1 in targeting cells. METHODS: ANG-1 cDNA was obtained from human spleen by RT-PCR and was inserted into AAV vectors to form rAAV ANG-1, the virus stocks in high titer were harvested. The rAAVANG-1 and rAAV GFP were transferred into pig mesenchymal stem cells and the expression of ANG-1 was detected by Western blot. RESULTS: The cloned ANG-1 cDNA was 1515bp in length which was in accordance with that reported previously. Titration of rAAVANG-1 stock was 9 X 10(11)v.g/ml. The expression of ANG-1 gene was detected in transfected cells. Forty-eight hours after rAAV GFP was transfected into mesenchymal stem cells, 55% cells expressed GFP. CONCLUSION: The constructed rAAV ANG-1 vector has successfully transfered and expressed in pig mesenchymal stem cells.

[Create a standard mini-swine model of chronic ischemic myocardium by thoracoscopy].

OBJECTIVE: To create a standard mini-swine model of chronic ischemic myocardium by endoscopy for the research of gene transfer and stem cell. METHODS: Twenty-three male China experimental minipigs were used, aged from 8 to 11 months with a mean of (9.3 +/- 1.8) months and weighed from 20 to 30 kg with a mean of (29.3 +/- 4.3) kg. The myocardial ischemia was established by gradual occlusion of the left circumflex coronary artery (LCX) with an Ameroid constrictor. The Ameroid constrictor was implanted around LCX by endoscopy. Selective coronary angiography, electrocardiogram and Echo-Doppler study were performed perioperatively to evaluate the degree of stenosis. RESULTS: Chronic ischemic myocardial models were successfully generated in 20 of 23 swine by full-endoscopy. Ameroid constrictors were placed at the LCX accurately. Three swine died of anesthetic accident, cardiac arrhythmia at secondary coronary angiography, and pulmonary infection within 6 weeks after operation respectively. Operation time was 25 to 65 min with a mean of (46 +/- 9) min. The blood loss was 30 to 60 ml with a mean of (55 +/- 12) ml. Six weeks later, coronary angiography revealed the total occlusion and partial stenosis (> 85%) of the LCX occurred in 7 and 13 swine respectively. Cardiac systolic and diastolic dysfunction were found in all swine. The ejection fraction value was (65.0 +/- 6.3)% before operation and (41.0 +/- 9.3)% after operation (P = 0.008). The fractional shortening value was (36.2 +/- 4.3)% before operation and (34.2 +/- 2.3)% after operation (P = 0.027). CONCLUSION: The endoscopic surgery is a less invasive way to create a standard mini-swine model of chronic ischemic myocardium with effective results.