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Objective To investigate the mechanism of rapamycin inhibiting the differentiation and proliferation of newborn porcine pancreatic adult stem cells, and to explore the therapeutic methods that may effectively reduce the side effects of rapamycin. Method Porcine NPCCs were treated with rapamycin alone or in combination with IGF-Ⅱ, and the caspase-3 and [ 3 H ]-thymidine uptake assays were performed to detect apoptosis and proliferation. The expression of insulin, PDX-1, NeuroD/Beta2, and Foxo1, a downstream transcription factor of IGF-Ⅱ, were analyzed by RT-PCR and Western blot to evaluate the differentiation ability of pancreatic adult stem cells. Results The NPCCs treated with rapamycin inhibited the proliferation ofβ-cells, increased apoptosis, reduced insulin secretion, inhibited the expression of PDX-1 and NeuroD/Beta2, and decreased the expression of IGF-Ⅱ. Foxo1 expression and induction of Foxo1 from the cytoplasm to the nucleus of the ectopic. The combined treatment of rapamycin and IGF-Ⅱcan reduce the side effects of rapamycin, inhibit the decrease ofβ-cell number and insulin content, repair the expression of insulin, PDX-1, NeuroD/Beta2, inhibit Foxo1 expression and intracellular ectopic. Conclusion Aberrant expression of IGF-Ⅱ and Foxol genes is the key inducing factor of rapamycin inhibiting the proliferation and differentiation of NPCCs, and IGF-Ⅱtreatment can effectively reduce the side effects of rapamycin on NPCCs differentiation.
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Objective To study the effects of serum C-type natriuretic peptide (CNP) ,insulin-like growth factor II (IGF-Ⅱ ) , endothelin (ET) ,neuron-specific enolase (NSE) and S100B protein(S100B) on the prognosis of the patients with traumatic brain injury .Methods A total of 110 patients with craniocerebral trauma admitted in our hospital from January 2016 to January 2017 se-lected as the craniocerebral trauma group and further divided into the mild ,moderate and severe craniocerebral trauma groups ac-cording to the Glasgow Coma Scale (GCS) .Then the levels of serum CNP ,IGF-Ⅱ ,ET ,NSE and S100B in all cases were analyzed by enzyme-linked immunosorbent assay (ELISA) .Their influence on the prognosis of the patients with craniocerebral trauma and the correlation among various indicators were analyzed .Results The levels of CNP and IGF-Ⅱat admission in the craniocerebral trauma group were significantly decreased ,while the levels of ET ,NSE and S100B were significantly increased ,the difference com-pared with the control group was statistically significant (P<0 .05) .Serum CNP and IGF-Ⅱlevels in the death group ,plant survival group and disabled group were significantly decreased .The difference was statistically significant (P<0 .01) .Serum CNP and IGF-Ⅱlevels in the moderate and severe craniocerebral trauma groups were gradually increased with the disease course progress ,while serum ET ,NSE and S100B levels were gradually decreased with the disease course progress ,the difference was statistically signifi-cant(P<0 .05) .In the patients with craniocerebral trauma ,the positive correlation existed between CNP and IGF-Ⅱ ,between ET and S100B ,between ET and NSE ,and between NSE and S100B(P<0 .01) ,while the negative correlation existed between IGF-Ⅱand ET ,between IGF-Ⅱ and S100B ,between CNP and ET ,and between IGF-Ⅱand NSE (P<0 .01) .Conclusion Serum CNP , IGF-Ⅱ ,ET ,NSE and S100B are correlated to the severity of craniocerebral trauma ,which has a higher clinical application value for judging the disease condition ,evaluating the prognosis in cradiocerebral trauma .
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Objective To investigate the expression of insulin-like growth factor Ⅱ mRNA binding protein 3 (IMP3) in non-muscle invasive bladder cancer and its relationship with the tumor recurrence and progression.Methods IMP3 protein expression was detected by immunohistochemistry in 130 cases of nonmuscle invasive bladder cancer specimens who underwent transurethral resection the first time at Beijing Chao-Yang hospital,from October 2010 to October 2013.Besides,we analyzed 20 cases of muscle-invasive samples and 20 benign tissues adjacent to cancer as control.The 130 patients were followed up by telephone and other methods.According to the UICC-TNM standard.Survival analysis was calculated by using the Kaplan-Meier method,and the difference in survival curves was analyzed by using the log-rank test.For multiple analyses,The Cox proportional hazards regression model was used.Results The positive expression rate of IMP3 in 130 patients with non-muscle invasive bladder cancer was 59.2％ (77/130),of which 30.0％ (39/130) was weak expression,29.2％ (38/130) was strong.However there was 80.0％ (16/20) in muscle-invasive specimens,of which 20.0％ (4/20) was weak,60.0％ (12/20) was strong (P =0.011).IMP3 was not detected in all benign tissues adjacent to cancer (P ＜0.001).All the 130 patients were followed-up for 5 to 69 mnonths,45 cases experienced disease recurrence,20 patients had progressed and 12 cases died.IMP3 expression was significantly related to higher tumor stage (P ＜ 0.001),high tumor grade (P =0.014),and tumor recurrence (P =0.003).Kaplan-Meier plots and log-rank tests showed that patients with IMP3-positive tumors had a lower disease-free survival (P =0.002) and progression-free survival rate (P =0.010) than those with IMP3-negative tumors.In the multivariable Cox analysis,we found that IMP3 protein was an independent predictor of disease-free survival (P =0.010) in non-muscle invasive urothelial carcinoma of bladder.Conclusions IMP3 was not expressed in benign tissue adjacent to cancer,whereas highly expressed in bladder cancer,and high IMP3 expression is an independent prognostic factor in NMIBC that can identify the patients with a high potential to relapse.
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Objective To explore the role of insulin-like growth factor-2/insulin-like growth factor1 receptor/insulin receptor substrate-1 (IGF2/IGF1R/IRS1) signal pathway inducing the chemoresistance of epidermal growth factor receptor 2 (ErbB2) positive breast cancer cells to Herceptin.Methods Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assay were used to determine the expression levels of IGF2,IGF1 R,and IRS1.The direct targets of miR-126 were validated by dual-luciferase reporter gene assay.In SKBR3/pool2 cells,IGF1 R activity was reduced by an inhibitor of IGF1 R,and IRS1 was knocked-down by shRNAs.Furthermore,3-(4,5-dimenthylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed to evaluate the sensitivity of these treated cells to Herceptin.Results IGF2,IGF1 R,and IRS1 were significantly higher expressed in SKBR3/pool2 cell compared to that in SKBR3 cell.Western blot assay showed that IGF2/IGF1R/IRS1 was activated in SKBR3/pool2 cells.Bioinformatics analysis combined with luciferase activity suggested that miR-126 directly targeted IRS1.MTS results demonstrated that the chemosensitivity to Herceptin of SKBR3/ pool2 cells with inhibitor of IGF1R or shRNAs targeting IRS1 or overexpressing miR-126 was significantly reduced.Conclusions IGF2/IGF1R/IRS1 signal pathway confers to the chemoresistance of ErbB2 positive breast cancer cells to Herceptin.
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Objective:To detect the methylation status of insulin-like growth factor Ⅱ(IGF-Ⅱ)gene promoter P3 in salivary pleo-morphic adenoma(SPA).Methods:The methylation of IGF-Ⅱ gene promtor P3 was examined in 26 cases of salivary pleomorphic adenoma with adjacent normal salivary gland tissues,1 0 cases of salivary gland malignant tumor (except malignant pleomorphic ade-noma)and 1 0 cases of normal salivary gland tissue by nested methylation specific polymerase chainreaction(nMSP),1 0 samples(3 SPA and controls,2 malignance and controls)were examined by pyrosequencing.Results:9 out off the 26 SPA showed lower level methylation oevel of IGF-II gene promoter P3.Normal salivary gland and salivary gland malignant tumor tissue did not.Conclusion:IGF-II gene promoter P3 with low level of methylation may play a role in the development of SPA.
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Objective To discuss the expressions and clinicopathologic significances of insulin-like growth factor Ⅱ mRNA binding protein 3 (IMP3)in squamous intraepithelial lesion (SIL)and cervical squa-mous cell carcinoma (CSCC)before and after the therapy of radiation and chemotherapy.Methods The expressions of IMP3 in 80 cases of CSCC,90 cases of SIL (60 cases of HSIL,30 cases of LSIL)and 30 cases of cervicitis were detected by immunobistochemistry.The relations between IMP3 and clinicopathological cha-racteristics of CSCC were analyzed.Results The expression rates of IMP3 in CSCC,HSIL,LSIL and cervici-tis were 86.3%(69 /80),78.3%(47 /60),33.3%(10 /30)and 0(0 /30),and the difference among the four groups was statistically signicant (χ2 =87.01,P <0.01).The positive expression rate of IMP3 declined by radiation or chemotherapy (60.0% vs.85.0%,χ2 =5.79,P =0.013).The expression of IMP3 was related with lymph node metastasis (χ2 =3.97,P =0.046),differentiated degree (χ2 =5.95,P =0.018),clinical stage (χ2 =5.82,P =0.016)and invasion depth (χ2 =5.73,P =0.017).There was nothing to do with age (χ2 =0.11,P =0.745).Conclusion IMP3 expresses excessively in CSCC,and is associated with pathologi-cal grade and invasion progress.Radio-chemotherapy can reduce the expression of IMP3.
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Objective To investigate the alternation of insulin -like growth factor -Ⅱ( IGF-Ⅱ) gene promoter P4 methylation status in hepatocellular carcinoma ( HCC) and explore its relationship with expression of P4 mRNA levels.Methods Liver specimens of 43 patients with HCC and normal liver specimens of 9 control patients were collected in operation .Tissue DNA and total RNA were extracted from these specimens .IGF-Ⅱ P4 methylation status and P4 mRNA expression levels were detected .Results (1)The incidence of IGF -ⅡP4 methyl-ation in HCC group was significantly lower than that in normal liver specimens (16.28%vs 88.89%,χ2 =19.12,P<0.01).(2)The expression level of IGF -ⅡP4 mRNA in HCC group was significantly higher than that that in normal liver specimens[(0.96 ±0.74) vs (0.25 ±0.19),t=5.48,P<0.01].(3)In HCC group,the IGF-Ⅱ P4 mRNA expression level with hypomethylation gene was significantly higher than that without hypomethylation gene [(1.18 ± 0.76) vs (0.32 ±0.27),t=5.28,P<0.01].Conclusion The hypomethylation alternation of IGF -Ⅱ P4 gene promoter which is accomplished by up -regulate P4 mRNA expression has a close relationship with HCC .
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Metabolic syndrome (MS) and breast cancer are common diseases of women. Triple negative breast cancer (TNBCs) is one type of breast cancer, which is of much attention in recent years. Important components of MS include central obesity, high blood sugar, high triglycerides and low level of high-density lipoprotein (HDL-C), which increased the inci-dence risk of TNBCs. Common biomarkers of MS including insulin, adiponectin and leptin play an important role in the oc-currence and development of breast cancer, especially TNBCs. Insulin-like growth factor-IImRNA binding protein 3 (IMP3, an oncofetal protein) may be TNBCs’new invasive cancer biomarkers. In this paper, the research progress on the relation-ship between MS and TNBCs is reviewed.
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Objective To detect the expression of insulin-like growth factor Ⅱ mRNA-binding protein 3 (IMP3) and p16 in cervical carcinoma and investigate their clinical significance.Methods Immunohistochemical Envision method was used to detect the expression of IMP3 and p16 proteins in 67 cases of carcinoma of the uterine cervix,58 cases of cervical intraepithelial neoplasia (CIN) and 15 cases of normal cervical epithelium.Results IMP3 was mainly dedected in the cytoplasm,whose positive expression rate was 85.07％ (57/67) in cervical carcinoma,12.07％ (7/58) in CIN (x2 =66.32,P ＜ 0.05) and 0 (0/15) in cervical epithelium (x2 =44.38,P ＜ 0.05).No significance variations of the expression of IMP3 were observed in cervical adenocarcinoma and squamous cell carcinoma (x2 =0.35,P ＞ 0.05).p16 was found both in nucleus and cytoplasm of cervical carcinoma,whose expression rate was 83.58％ (56/67),higher than 41.37％ (24/58) in CIN (x2 =24.85,P ＜ 0.05) and 0 (0/15) in cervical epithelium (x2 =42.61,P ＜ 0.05).Conclusion Expression of IMP3 and p16 is high in cervical carcinoma,maybe can be used as a potential biomarker of metastasis and progression.
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Objective To study the expression of insulin-like growth factor-Ⅱ receptor(IGF-Ⅱ R) and its relationship with the prognosis of human epidermalgrowth factor receptor 2 (HER2) positive breast cancer.Methods The expression of IGF-Ⅱ R in 104 specimens of HER2 positive breast cancer was evaluated by immunohistochemistry.The expression was rated on 2 scales:negative and positive.The age,pathological type,tumor size,lymph node involvement,status of estrogen receptor(ER),and progesterone receptor(PR) of all the cases were reviewed.The relationship between the expression of IGF-Ⅱ R and the above pathological parameters were analyzed by statistical methods.The relationship between the expression of IGF-Ⅱ R and the disease-free survival and overall survival was studied.Results The positive expression rate of IGF-Ⅱ R was 43.3％ (45/104).The positive rate of IGF-Ⅱ R was significantly higher in the lymph node positive group than in the lymph node negative group (61.5 ％ vs 25.0％,P =0.000).No significant correlation was observed between the expression of IGF-Ⅱ R and age,pathological type,tumor size,status of ER,or PR.The disease-free survival and overall survival was significantly lower in IGF-Ⅱ R positive group than in IGF-Ⅱ R negative group.(The 5-year disease-free survival:62.6％ vs 84.7％,P =0.022; The 5-year overall survival:71.5％ vs 89.6％,P =0.024).Cox regressive analysis showed that the lymph node status was an independent risk factor of disease-free survival and overall survival,while the IGF-Ⅱ R was not.Conclusion The positive expression of IGF-Ⅱ R in HER2 positive breast cancer indicates poor prognosis.The expression of IGF-Ⅱ R may be regulated by insulin-like growth factor-Ⅱ (IGF-Ⅱ).
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Objective To study the change and clinical significance of insulin-like growth factor Ⅱ (IGF-Ⅱ) in patients with bone fractures accompanied by craniocerebral trauma.Methods The level of IGF-Ⅱ was detected by ELISA in 40 patients with bone fractures accompanied by craniocerebral trauma (combined with craniocerebral trauma group) and 40 patients of simple fracture (simple fracture group) after injury 1,3,7,14 d.Forty healthy person was as control group.The rate of porosis after injury 4,8,12 weeks was compared.Results The level of IGF-Ⅱ in combined with craniocerebral trauma group after injury 1,3,7,14 d was (4.15 + 1.38),(7.69 ± 2.40),(11.97 ± 3.74) and (14.08 ± 4.69) μg/L,simple fracture group was (2.56 ± 0.85),(3.70 ± 1.16),(4.96 + 1.55) and (7.52 ± 2.51) μg/L,control group was (2.10 ±0.70) μg/L.The level of IGF-Ⅱ in combined with craniocerebral trauma group and simple fracture group was significantly higher than that in control group (t =8.363,14.121,16.403,15.968;2.636,7.495,10.635,13.171,P ＜ 0.05).The level of IGF-Ⅱ in combined with craniocerebral trauma group was significantly higher than that in simple fracture group (t =6.187,9.449,10.950,7.799,P＜ 0.05).The rate of porosis after injury 4,8,12 weeks in combined with craniocerebral trauma group was 22.5％ (9/40),77.5％ (31/40),100.0％(40/40),significantly higher than that in simple fracture group [5.0％ (2/40),27.5％ (11/40),47.5％ (19/40)] (x2 =5.165,29.463,28.475,P ＜ 0.05).Conclusion The serum IGF-Ⅱ may become a new factor promoting the healing of fracture and this study provide new ideas for the treatment of fracture.
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Objective To observe the effects of insulin-like growth factor-2 (IGF2) in the course of mouse embryon-ic stem cells induced to differentiate into islet-like cells. Methods Mouse ES cells were induced to differentiate into islet-like cells in vitro. The expression of islet specific markers was tested by RT-PCR and immunofluorescence assay. RT-PCR/RFLP was used to test the imprinted genes IGF2 parental expression in cells at different stages. Results Islet specific mark-ers were expressed in differentiated cells, such as insulin, glucagon and C-peptide. PCR-RFLP showed that imprinted genes IGF2 derived from embryonic stem cells were biallelic expression and loss of imprinting. Conclusion Gene imprinting sta-tus of IGF2 was changed in differentiated cells in vitro.
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Objective To study the expression changes of placental insulin-like growth factor Ⅰ and Ⅱ (IGF-Ⅰ and IGF-Ⅰ) between pregnancies following the in vitro fertilization and embryo transfer (IVF-ET) and those conceived spontaneously.Methods The placenta were collected from 49 cases of pregnancies after IVF-ET(case group) and 49 cases of pregnancies who were normal (control group).The expressions of IGF-Ⅰ and IGF-Ⅱ mRNA in placenta were detected by reverse transcription-polymeras chain reaction (RT-PCR).The levels of IGF-Ⅰ and IGF-Ⅱ protein were determined by immunohistochemistry.Results The mRNA levels of the placental IGF-Ⅰ and IGF Ⅱ were 0.30 ±0.13 and 0.28 ±0.04 in the IVF-ET group,and 0.65 ±0.10 and 0.91 ±0.26 in the control group.The protein levels of the placental IGF-Ⅰ and IGF-Ⅱ were 0.26 ±0.04 and 0.29 ± 0.05 in the IVF-ET group,and 0.32 ± 0.07 and 0.34 ± 0.04 in the control group.The mRNA and protein expressions of IGF-Ⅰ and IGF-Ⅱ in human placenta were significantly decreased in IVF-ET group compared to control group (P ＜ 0.05).The incidence rate of low birth weight infant in IVF-ET group was significantly higher than control(P ＞ 0.05).Conclusions Expressions of placental IGF-Ⅰ and IGF-Ⅱ in the IVF-ET group did not affect fetal growth and development.
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Object To investigate the correlation of IGF-2 exon 9 CpG island methylation and the expression of IGF-2,and further explore the mechanism of Wilms tumor. Methods The IGF-2 exon 9 methylation status and IGF2 gene expression in 42 cases of Wilms tumor and corresponding normal tissues were detected by using methylation-sensitive restriction enzyme PCR and allele-specific IGF2 gene expression analysis.Results IGF-2 exon 9 methylation rate was 15% in W ilms tumor group, and 97% in normal tissue group. The difference was significant (P < 0.01), and the difference in pathological type was also significantly different (P < 0.01). Exon 9 unmethylated tissues showing biallelic expressions of IGF-2 were significantly higher than that of methylated tissues (P < 0.05). Conclusion IGF-2 exon 9 unmethylation may be the cause of biallelic expression of IGF, which contritues to tumorigenesis in Wilms tumor.
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ObjectiveTo investigate the expressions and significance of insulin-like growth factor Ⅱ and basic fibroblast growth factor in adrenal disease. MethodsThe experiment was divided into groups of adrenal pheochromocytoma,corticomedullary hyperplasia,cortical adenoma,normal adrenal tissue.Every group was 15 cases.Expression of IGF-Ⅱ and bFGF were detected by immunohistochemistry in adrenal disease. ResultsThere was a significant difference among the four groups(all P＜0.05).IGF-Ⅱ and bFGF expression was higher in adrenal pheochromocytoma tissue than that of corticomedullary hyperplasia and cortical adenoma.In the normal adrenal gland group was low expression or no expression.In each group,IGF-Ⅱ and bFGF protein expression was positively correlated(all P＜0.05).There also was a positive correlation between IGF-Ⅱ and bFGF positive cell and blood pressure in adrenal pheochromocytoma,corticomedullary hyperplasia and cortical adenoma(all P＜0.05). ConclusionIGF-Ⅱ and bFGF protein were closely related to the occurrence and development of human adrenal corticomedullary hyperplasia,cortical adenoma,pheochromocytoma,and played a role in hypertension caused by them.At the same time there was a synergistic effect between the IGF-Ⅱ and bFGF protein in adrenal disease.
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Objective To observe the expressions of serum insulin-like growth factor (IGF)- Ⅰ ,Ⅱ and IGF binding protein (IGFBP) 3, 5 and to explore the clinical significances in patients with clear cell carcinoma of kidney. Methods Enzyme-linked immunosorbent assay (ELISA) methods were adopted to examine serum expressions of IGF-Ⅰ , Ⅱ and IGFBP 3, 5 in 40 cases with clear cell carcinoma of kidney (renal carcinoma group) and 16 cases with hydronephrosis (control group) from May 2007 to December 2009. Results IGF- Ⅰ , Ⅱ and IGFBP 3,5 in renal carcinoma showed higher expressions before operation (985. 7 μg/L, 1154.0 μg/L,46.6 μg/L and 9.6 μg/L, respectively)than after operation (431.4 μg/L, 632.6 μg/L, 26.7 μg/L, and 6.7 μg/L, respectively, all P＜0. 05 ～0.01). There were no significant differences in those indexes between pre- and post- operation in control group (P＞ 0. 05). Conclusions There are high expressions of serum IGF-Ⅰ , Ⅱ and IGFBP 3, 5 in renal carcinoma patients, and IGF- Ⅱ has clinical significance in diagnosis.
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Objective To investigate the effect of GH on proliferation of pancreatic cancer cells and observe the features of IGF-IGFBP3 pathway in the host after GH administration. Methods Pancreatic cancer cells (SW-1990,PANC-1 and P3) during exponential growth stage were harvested and cultured in medium containing growth hormone (50 ng/ml). After 24, 48 and 72 hours, cells were counted using a Coulter Counter. Thirty-five Athymic nude Balb/c mice were inoculated with SW-1990cells. When tumors became palpable after inoculation, animals were randomized to receive GH points (1 h, 2 h, 6 h, 24 after the last injection), plasma samples were gathered for subsequent ELISA determination and liver was rapidly incised for immune blotting analysis. Results The results revealed that GH stimulated cell growth in vitro. GH elevated levels of IGF-Ⅰ , Ⅱ at the 1st , 2nd , 6th hour after the last injection. GH augmented the expression of IGFBP3 in the liver of the host in vivo (1 h, 2 h, 6 h, 24 h, respectively). Conclusion Such proteins as IGF- Ⅰ and Ⅱ might be associated with mechanism of last effect of GH on tumor host. The up-regulation of IGFBP3 by GH administration in the host may help to explain the phenomena that GH doesn't accelerate growth of pancreatic tumor in vivo.
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Objective To study the expression of the imprinted gene H19 and IGF-Ⅱ in newborn placenta,and to discuss its influence on the birth body mass of the neonate. Methods The fresh placental tissues from full-term newborn (without trimester of pregnancy complica-tion and placenta and funic abnormality) with normal,high and low birth body mass (12,10 and 8 samples respectively)were collected. The expression of imprinted gene H19 and IGF-Ⅱ mRNA in the placenta were estimated by reakime fluorescence quantitative PCR Results The ex-pression of H19 mRNA in the placenta was negative correlation to the birth body mass (r =-0.403,P = 0.027).The expression of of IGF-H mRNA was positive correlated to the birth body mass (r = 0.444,P = 0.014). The H19 mRNA expression level in the high birth weight neonates (0.21 0.31) was significantly lower than that in the low birth body mass neonates (1.51 2.04)(P= 0.013). But the expression level of IGF-Ⅱ mRNA in the high birth body mass neonates (2.67±3.41) was significantly higher than that in the low birth body mass neonates (0.39±0.33)(P =0.013). Conclusion The expression of H19 and IGF-Ⅱ mRNA was significantly different in the placenta of normal,high and low birth body mass newboms. These two genes may be related to the birth body mass,and there may be some realation-ship between these two genes.
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Objective To investigate the clinical significance of changes of serum insulin-like growth factor-Ⅱ(IGF-Ⅱ),carbohydrate antigen 19-9(CA19-9)and alpha fetoprotein(AFP)levels after intervention and percutaneous ethanol injection therapy in patients with primary hepatic cancer.Methods Serum levels of IGF-Ⅱ,CA19-9 and AFP(with RIA)were repeatedly determined in 57 patients with primary hepatic cancer before intervention therapy,1 month after intervention and percutaneous ethanol injection therapy and 6 months after intervention and percutaneous ethanol injection therapy as well as in 42 controls.Results Before intervention therapy,serum leveh of IGF-Ⅱ,CA19-9 and AFP in the patients were significantly higher than those in the controls(P＜0.01).One month after intervention and percutaneous ethanol injection therapy,all the serum levels were near to normal.Six months later,the levels in the patients without recurrence remained normal.However,the levels in the 10 patients with recurrence returned to those before intervention therapy again.Conclusion Changes of serum IGF-Ⅱ,CA19-9 and AFP levels are closely related to the tumor burden and may reflect the presence of recurrence.
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AIM: To construct the shuttle plasmid vector for thymidine kinase (tk) and EGFP fusion protein gene driven by IGF - Ⅱ P3 promoter, and investigate the specific killing effect of the HSV - tk/GCV system on hepatocellular carcinoma(HCC) cells in vitro. METHODS: Recombinant shuttle plasmid vector was constructed by techniques of genetic recombination and screening, and identified by restriction digestion and sequencing analysis. Then the recombinant shuttle plasmid was transfected into HepG2 and HeLa cells by techniques of lipofectamine transfection and its expression was detected by fluorescence microscope and RT -PCR. Cell killing after ganciclovir(GCV) application was determined by MTT. RESULTS: Identification of pDC316 -tkEGFP- P3 by enzyme digestion and sequencing analysis showed that the length, inserted location and direction of the target genes which were inserted into the recombinant were correct. It was found that enhanced green fluorescence protein could only be seen in HepG2 cells, but not in HeLa cells. The results of RT -PCR showed that only two bands could be seen in the samples of pDC316 -tkEGFP- P3 transfected HepG2 cells. The MTT test showed the selective cytotoxicity of GCV to the transfected HepG2 cells. CONCLUSION: The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF - Ⅱ P3 promoter has been constructed successfully and its specific expression in HepG2 cells provided a sound basis for targeted gene therapy for HCC.