3.Construction of Formin-like 2 specific small hairpin RNA expressing plasmid and its effect on proliferation in SW620
Xiling ZHU ; Li LIANG ; Yanqing DING
Clinical Medicine of China 2008;24(6):513-517
Objective To construct the specific small hairpin RNA(shRNA)plasmids for Formin-like 2(FMNL2)gene,and to observe its effect on proliferation of SW620.Methods Plasmids FMNL21 shRNA and plasmids FMNL22 shRNA complimentary to the sequence responsible for the function domain of human FMNL2 were constructed and transfected into SW620,respectively.The inhibition rates of FMNL2 expression were detected by re-al-time PCR after transfection with PGenesil-FMNL21 and PGenesil-FMNL22,respectively.The expression of FMNL2 was detected by real-time PCR at 12,24,48,72 and 96h after transfection.At 48 h and 72 h after transfection,the proliferation of SW620 was detected using MTT assay.Resuits The transfection rate in SW620 was(60.8±2.8)%for PGenesil-FMNL21,(58.7±2.9)%for PGenesil-FMNL22,and(62.6±1.7)%for PGenesil-HK(P>0.05).The inhibition rate of FMNL2 expression after transfection with PGenesil-FMNL21 was 77.1%,which was the highest.The inhibition rates of FMNL2 expression were 13.5%,57.3%,80.3%,62.4%,and 33.2%at 12,24,48,72and 96h after transfection respectively.The proliferat capability of SW620 was obviously lower than that of control cells(P<0.05).Conclusion Both plasmids FMNL21 shRNA and plasmids FMNL22 shRNA can inhibit the expression of FMNL2,of which Pgenesil-FMNL21 execs the greatest role.FMNL2 could be related to the proliferation of SW620.
4.The expression and clinical significance of formlin-like 2 in colorectal carcinoma
Xiling ZHU ; Li LIANG ; Yanqing DING
Clinical Medicine of China 2008;24(5):401-404
Objective To analyze the relationship between the expression of formlin-like 2(FMNL2)and colorectal carcinoma metastasis through the detection of the expression of FMNL2 in colorectal carcinoma tissue and paracancerous tissue.Methods ①Immunohistochemistry was used to detect the expression of FMNL2 in 175 cases of paraffin wax specimens(including 30 cases of normal mucosa,25 cases of adenoma,75 cases of colorectal carcinoma and 45 cases of metastatic tumors).②Real time-PCR was used to detect FMNL2 mRNA expression in 32 cases of fresh specimens of carcinoma and 32 cases of paracancerous tissue.Results The positive detection rate of FMNL2 was higher in colorectal carcinoma tissue than in paracancerous tissue(P=0.003),higher in lymphatic metastasis than in primary carcinoma tissue(P=0.037),and was aslo higher in metastatic carcinoma tissue than in non metastatic carcinoma tissue(P=0.011).The expression of FMNL2 mRNA was not related to the infiltration and differentiation of carcinoma tissue but its concentration in metastatic carcinoma tissue was 2.5 times higher than in non metastatic carcinoma tissue.Conclusion FMNL2 gene may play an important role in the metastasis of colorectal carcinoma.
5.Mechanism of vascular endothelial growth factor up-regulating intercellular adhesion molecule-1 via protein kinase C/nitric oxide pathway in the retina of diabetic rats
Xiaoling ZHANG ; Yanning DING ; Yuan ZHU ; Liang WEN
Journal of Xi'an Jiaotong University(Medical Sciences) 1981;0(02):-
Objective To investigate how vascular endothelial growth factor(VEGF) up-regulates intercellular adhesion molecule-1 via protein kinase C(PKC)/ nitric oxide(NO) pathway in the retina of diabetic rats.Methods All the rats were divided into 4 groups: normal,diabetes,diabetes+PKCI and control groups.Diabetes was induced by an intraperitoneal injection of STZ.PKC inhibitor GF109203X was injected intravitreally after 5 months of streptozotocin induced diabetes.NO was determined by nitrate reductase method.VEGF and intercellular adhesion molecule-1(ICAM-1) were measured by Western blot.Results VEGF and NO expressions increased obviously in the retina of diabetic rats compared with those in the normal group(P
6.Experimental Study on the Prevention of Skin Flap Necrosis after Modified Radical Mastectomy in Breast Cancer by Procaine
Liang MING ; Zhaoyong CHEN ; Xian DING ; Shumin DONG ; Min ZHU
Journal of Medical Research 2006;0(09):-
Objective To evaluate the effect of procaine on prevention of skin slap necrosis after modified radical mastectomy in breast cancer.Methods 106 breast cancer patients were randomly divided into treatment and control groups.In the treatment group,procaine(1% 100ml diluted in 300ml mormal saline,42℃)were compressed under the skin flap in operation,the control group received same amount of normal salin instead.The rates and sizes of skin flap necrosis were obeserved and compared.Results There were 13 cases suffured from skin flap necrosis among 106 breast cancer cases who received surgery operation.The rate of the flap necrosis in treatment group was5.76%(3/52),and the control group was 18.51%(10/54),there was significant difference(P
7.Galectin-1 expression in human colorectal carcinoma and its clinical significance.
Xi-ling ZHU ; Li LIANG ; Yan-qing DING
Journal of Southern Medical University 2007;27(9):1331-1334
OBJECTIVETo investigate the correlation between galectin-1 expression and the biological behaviors of human colorectal carcinoma.
METHODSSP immunohistochemistry was used to detect the expression of galectin-1 in 158 paraffin-embedded specimens including 30 normal mucosa, 25 adenoma, 65 colorectal carcinoma and 38 metastatic tumor specimens. Real-time RT-PCR was used to detect galectin-1 mRNA expression in 32 fresh specimens of colorectal carcinoma and normal mucosa.
RESULTSThe positive expression level of galectin-1 was significantly different between normal mucosa, adenoma, colorectal carcinomas and metastatic tumors, with positivity rate of 0, 8%, 66% and 86%, respectively (P<0.05). Galectin-1 expression in moderately or well differentiated colorectal carcinomas was significantly lower than that in poorly differentiated ones (P=0.031), and its expression in invasive carcinomas was significantly higher than that in non-invasive carcinomas (P=0.000). Galectin-1 expression in colorectal carcinomas was significantly related with lymph node metastasis (P=0.004). In poorly differentiated colorectal carcinomas, the expression of galectin-1 mRNA was about 2.27 times that in moderately or well differentiated colorectal carcinomas (P=0.00); galectin-1 mRNA expression in invasive carcinoma was 1.98 times that in non-invasive carcinoma (P=0.002). In tumors with lymph node metastasis, galectin-1 mRNA expression was 1.42 times that in tumors without metastasis (P=0.018).
CONCLUSIONGalectin-1 can be involved in the development and progression of colorectal carcinoma, and may relate to the infiltration, differentiation and lymph node metastasis of colorectal carcinoma.
Colorectal Neoplasms ; genetics ; pathology ; Galectin 1 ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Intestinal Mucosa ; cytology ; metabolism ; pathology ; Neoplasm Invasiveness ; Neoplasm Metastasis ; genetics ; Polymerase Chain Reaction ; RNA, Messenger ; genetics ; metabolism
8.RNA interference affects the feak-headbox 3a gene expression in myoblast cell line L6
Jie DING ; Bingsheng LIANG ; Zhifeng DA ; Zhixiang ZHU ; Jian WEI ; Yingwei JIA ; Yong FENG
Chinese Journal of Tissue Engineering Research 2013;(33):5974-5980
BACKGROUND:Recent studies found that some factors play important role in the process of denervated muscle atrophy, especial y the feak-headbox transcription factor, is the key element to regulate the denervated muscle atrophy.
OBJECTIVE:To investigate the effect of RNA interference on inhibiting feak-headbox 3a gene expression in vitro.
METHODS:The myoblast cel line L6 were cultured in the 6-wel cel culture plates, then pEGFP-N1 and smal
interfering RNA recombinant plasmid with the same ratio was transfected under the Lipofectamine2000 mediation to optimize the transfection efficiency of the detection system;2μg smal interfering RNA recombinant plasmid of feak-headbox 3a gene were transfected with myoblast cel line L6 for 48 and 72 hours.
RESULTS AND CONCLUSION:At 48 hours after pEGFP-N1 and siRNA recombinant plasmid transfection, a large number of bright green fluorescent displayed under fluorescence microscope with higher transfection efficiency. Real-time quantitative PCR analysis showed that there were significant differences in the sequences of feak-headbox 3a-Ⅰ, feak-headbox 3a-Ⅱ, feak-headbox 3a-Ⅲ, feak-headbox 3a-Ⅳ on feak-headbox 3a mRNA when compared with the control group at 48 and 72 hours after trasfection (P<0.05), and the inhibition effect was more significant at 72
hours after transfection when compared with that at 48 hours after transfection. Western Blot gray analysis showed that there were significant differences in sequences of feak-headbox 3a-Ⅰ, feak-headbox 3a-Ⅱ, feak-headbox 3a-Ⅲ,
feak-headbox 3a-Ⅳ on feak-headbox 3a mRNA when compared with the control group at 48 and 72 hours after
trasfection (P<0.05), and the inhibition effect was more significant at 72 hours after transfection when compared with that at 48 hours after transfection, which was same with the effect on mRNA level. RNA interference in vitro can
significantly inhibit the fork-head transcription factor feak-headbox 3a gene expression, and the inhibition effect of feak-headbox 3a gene smal interfering RNA recombinant plasmid transfected with the sequence on the mRNA and protein level of feak-headbox 3a is not clear, which can provide new idea for the gene therapy of RNA mediated denervated skeletal muscle atrophy.
9.Effect of gene silence of CD40 by small interfering RNA on balance between Th1/Th2 and Th17/Treg in rats with experimental autoimmune myocarditis
Guoyu DING ; Bo HAN ; Mei ZHU ; Rongjun ZHANG ; Ling GAO ; Jianjun ZHANG ; Yan LIANG
Chinese Journal of Applied Clinical Pediatrics 2015;(13):1011-1015
Objective To investigate how gene silence of CD40 by small interfering RNA(siRNA)influences the balance between Th1 / Th2 and Th17 / Treg in rats with experimental autoimmune myocarditis(EAM). Methods Lewis rats were divided into a normal - control group,EAM group,CD40 siRNA - therapy group and siRNA - control group by using random number table. EAM,CD40 siRNA - therapy and siRNA - control groups were immunized on day 0 and day 7 with purified porcine cardiac myosin to establish EAM,and CD40 siRNA was administered intravenously on day 7. The basic data of each group were observed,and the rats were executed on day 21 to study the pathology of myo-cardial tissues and the myocardial histopathology scores were calculated,and than the changes in electrocardiograms (ECG)and echocardiogram were recorded. Enzyme - linked immunosorbent assay(ELISA)was used to determine the levels of interferon(IFN) - γ,interleukin(IL) - 10,IL - 17 and transforming growth factor(TGF)- β in peripheral blood. Results (1)The overall incidence of EAM,CD40 siRNA - therapy and siRNA - control group was 100% ,and no rats died from day 0 to day 21.(2)There were only 2 premature rats in EAM and siRNA - control groups,and the ECGs of normal - control group and CD40 siRNA - therapy group were normal.(3)The echocardiogram of EAM,CD40 siRNA - therapy and siRNA - control group,displayed the left ventricular dilatation,hypertrophy of the left ventricle, the weakening of the left ventricular wall motion,and heart failure,and pericardial effusion was discovered. The left ven-tricular fractional shortening(LVFS)and the left ventricular ejection fraction(LVEF)of rats treated by CD40 siRNA were higher than those of rats in the EAM group[(46. 70 ± 8. 25)% vs(34. 28 ± 11. 81)% ,(80. 92 ± 11. 76)% vs (64. 03 ± 12. 60)% ,F = 0. 652,0. 234,all P ﹤ 0. 05].(4)Pathologic examination of the EAM group,CD40 siRNA therapy group and siRNA control groups,showed myocardial fiber fracture,myocardial tissue necrosis and inflammatory cell infiltration. But myocardial tissue pathological scores of the cardiac tissue of CD40 siRNA therapy group was lower than those of the EAM group(2. 10 ± 1. 07 vs 3. 40 ± 0. 72,F = 1. 290,P ﹤ 0. 05).(5)ELISA method examination re-vealed that the levels of IFN - γ,IL - 4,IL - 17 and TGF - β in EAM group were increased compared with those of nor-mal control group[(1 245. 55 ± 244. 56)ng/ L vs(501. 53 ± 18. 93)ng/ L,(78. 03 ± 10. 47)ng/ L vs(30. 77 ± 1. 74)ng/ L,(80. 82 ± 13. 33)ng/ L vs(27. 98 ± 3. 01)ng/ L,(156. 27 ± 12. 11)ng/ L vs(4. 33 ± 0. 79)ng/ L,t =7. 408,10. 764,9. 250,30. 608,all P ﹤ 0. 05]. However,the levels of IFN - γ and IL - 17 in CD40 siRNA therapy group were lower than those in the EAM group[(940. 62 ± 128. 40)ng/ L vs(1 245. 55 ± 244. 56)ng/ L,(60. 42 ± 12. 40) ng/ L vs(80. 82 ± 13. 33)ng/ L,t = 2. 704,2. 745,all P ﹤ 0. 05],while the levels of IL - 4 and TGF - β were higher [(97. 91 ± 13. 62)ng/ L vs(78. 03 ± 10. 47)ng/ L,(178. 84 ± 12. 10)ng/ L vs(156. 27 ± 12. 11)ng/ L,t = 2. 835, 3. 229,all P ﹤ 0. 05]. Conclusions The administration of CD40 siRNA markedly reduces myocardial inflammation of EAM rats and improves their cardiac function,which can be explained by Th1 - type and Th17 - type cytokines inhibi-ted,Th2 - type and Treg - type cytokines increased,and so then the imbalance of Th1 / Th2 and Th17 / Treg corrected.
10.Surgical repair of early bronchopleural fistula after pulmonary resection
Liang DUAN ; Xiaofeng CHEN ; Yuming ZHU ; Chang CHEN ; Hao WANG ; Wenpu TONG ; Jiaan DING ; Gening JIANG
Chinese Journal of Thoracic and Cardiovascular Surgery 2012;28(6):362-364
Objective Bronchopleural fistula (BPF) is a common but potentially lethal complication after pulmonary resection.Currently,there is still controversy over the appropriate management strategy for BPF,especially when pleural space contamination develops.The purpose of this study was to evaluate the efficacy and safety of surgical repair fistulas combined with pedicled muscle flaps coverage in patients with early BPF after pulmonary resection based on our experience with 23 cases.Methods The clinical data for 23 patients who underwent surgical repair of early BPF from January 1999 to December 2010 at our hospital were reviewed.Thirteen patients had undergone a prior pneumonectomy and 10 patients had undergone a prior lobectomy.BPF occurred from postoperative day 5 to40 (mean postoperative day 21 ).Nine patients had a contaminated pleural space.After BPF was clearly diagnosed,prompt closed pleural drainage was instituted,followed by surgical repair of BPF.Four patients underwent a direct suture repair of fistula,ten patients underwent stump revision and suture closure,seven patients underwent stump revision and bronchoplasty or carina plasty,and a pedicled muscle flap was sewn to the edges of the fistula in two patients.The stump was covered with various muscle flaps,including interostal muscle flap in five cases,latissimus dorsi muscle flap in ten cases,serratus anterior muscle flap in six cases,and erector spinae muscle flap in two cases.Postoperatively,the pleural space was routinely irrigated and drained.Results No intraoperative or early postoperative death occurred.Four patients developed severs complications,including respiratory failure in two cases,pulmonary embolism in one case,and empyema in one case.All four cases recovered well after treatment.The mean duration of hospitalization was 33 days (range 8 - 120 days ).Surgical repair of BPF was successful in 21 cases (91.3%) but failed for 2 patients..BPF recurrence developed in only one patient two years postoperatively due to stump recurrence.He died of extensive metastatic disease 2 years after BPF recurrence.Conclusion Excellent results can be achieved by early surgical repair combined with stump pedicled muscle flaps coverage in patients with BPF who can tolerate reoperation,even if they have a contaminaled pleural space.