The resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) has been brought into focus. COX-2 signal pathway was found to be closely related to EGFR signal pathway by recent researches, and there has been a growing interest to focus the researches on whether COX-2 pathway inhibition improves the efficacy of EGFR-TKIs in treating advanced NSCLC. In this review, we will illustrate recent advances of combined inhibition of EGFR and COX-2 signal pathways in NSCLC therapy.
Objective By using small interfering RNAs ( siRNAs) specific for spleen tyrosine kinase ( Syk) , to evaluate the role of Syk in maturation of bone marrow-derived dendritic cells. Methods The fragments of 21-23 bp siRNAs specific for mice Syk were chemo synthesized and transfected into the asthmatic murine bone marrow-derived dendritic cells ( BMDCs) by Lipofectamine 2000 transfection system for 48 hours. Then BMDCs were co-cultured with T cells from the normal mice spleen for 48 hours. The cytokines including IL-4, IL-13, IL-2 and INF-γin supernatant were detect by ELISA. The expression of Syk protein was measured by Western Blot to determine whether the Syk gene was silenced. Results The expression of Syk protein was obviously decreased in the siRNA-interference group. The secretions of IL-4 and IL-13 were significantly inhibited by siRNA interference ( P lt; 0. 05) , but the secretions of IL-2 and INF-γwere not interfered signficantly ( P gt;0. 05) . Conclusion Syk specific siRNA fragments can block the antigen presentation function of dendritic cells and block the activation and differentiation of T cells.
Objective To investigate the feasibility of detection of epidermal growth factor receptor ( EGFR) exon 19 deletions and exon 21 L858R mutations in pleural effusion fromnon-small-cell lung cancer ( NSCLC) patients by mutant enriched PCR assay. Methods The mutations of exon 19 and 21 of EGFR gene in pleural samples fromthirty NSCLC patients were analyzed using both the mutant-enriched PCR assay and the non-enriched PCR assay. Results Ten ( 33. 3% , 10/ 30) exon 19 deletions and five ( 16. 7% , 5/30) exon 21 L858R mutation were detected by the mutant-enriched PCR assay, while only 6 cases ( 20. 0% ) and 1 case ( 3. 3% ) were detected by the non-enriched PCR assay respectively. The difference of mutation detection rate of EGFR gene between the two methods was statistically significant ( P = 0. 032) . Mutations were detected in all of partial responders ( 2 /4) among the four patients who received gefitinib therapy. Conclusions Mutant-enriched PCR assay can detect EGFR exon 19 deletions and exon 21 L858R mutation in pleural effusion from NSCLC patients effectively, economically and accurately. It may be a valuable biomarker for gefitinib therapy in advanced NSCLC.
Objective To evaluate the effects of two different epidermal growth factor receptor tyrosine kinase inhibitors ( EGFR-TKIs) , Gefitinib and Erlotinib, on lung fibrosis induced by bleomycin.Methods Forty BALB/c female mice were randomly divided into four groups, ie. a control group( saline given orally and intratracheally) , a fibrosis group( saline given orally with bleomycin instillation) , a Gefitnib group( Gefitnib 20 mg/kg given orally with bleomycin instillation) , and an Erlotinib group ( Erlotinib25 mg/kg given orally with bleomycin instillation) . Bleomycin ( 3 mg/kg) was intratracheally instilled on the first day. Gefitinib or Erlotinib was given orally daily and normal saline as control. Then they were sacrificed by abdominal aortic bleeding 14 days after the bleomycin instillation. The left lung was stained with HE and Masson’s trichrome staining respectively for pathological examination. Total EGFR and phosphorylated EGFR were detected by immunohistochemistry. Hydroxyproline ( HYP) assay was performed in the right lung.Results Both Gefitinib and Erlotinib significantly reduced lung collagen accumulation and the content of HYP. Immunohistochemistry revealed that phosphorylation of EGFR in lung mesenchymal cells induced by bleomycin was inhibited. Furthermore, there was no difference between Gefitinib and Erlotinib in inhibiting lung fibrosis. Conclusion Our findings suggest that, in the preclinical setting, EGFR-TKIs may have aprotective effect on lung fibrosis induced by bleomycin.
Abstract:Objective To observe the expression of calcium-dependent proline-rich tyrosine kinase-2（Pyk2） in myocardium of rheumatic heart disease, the relationship between its role and cardiac fibrosis and clinical significance. Methods The blue myocardium collagen stain were analysed after Masson staining in 30 patients with rheumatic heart disease （RHD group） and 6 normal myocardium specimens （control group）. The contents of hyaluronic acid （HA）, laminin（LN） and type IV collagen（IV-C） were detected by radio-immunity method,and the expressions of Pyk2 protein and messenger ribonucleic acid（mRNA） were explored by immunohistochemistry methods and reverse transcriptase polymerase chain reaction （RT PCR）,then the correlations of these results were statistically analyzed. Results The contents of HA,LN and IV C in RHDgroup increased compared to control group（174.95±76.14μg/L vs. 70.06±15.63μg/L, 153. 86 ± 20. 72μg/L vs. 90.01±14. 11μg/L, 95. 26±7.66μg/L vs. 63. 21±10.62μg/L; P= 0.003, 0. 013, 0. 035）. The Pyk2 absorption and the ratio of Pyk2 mRNA/glyceraldehyde phosphate dehydrogenase （GAPDH） in RHD group were significantly higher than those in control group （0. 325 ± 0. 032 vs. 0.106±0.013, 0.870±0.085 vs. 0.573±0.042; P=0.048, 0.006）.There were positive correlativity between the expression of Pyk2 protein and HA, LN and IV-C （r=0. 611, 0. 743, 0. 829, P〈0. 01）, there were positive correlativity between the expression of Pyk2 mRNA and LN, IV-C （r=0. 794, 0. 766, P〈0.05）. Conclusion Pyk2 may play a key role in the proceeding of cardiac fibrosis in rheumatic heart disease by increasing collagen synthesis in myocardium.
Objective To construct gene silence adenovirus vector targeting both transglutaminase 2 （TG2） and Mer receptor tyrosine kinase （Mertk） synchronously and detect the gene silence function of it. Methods The interfering plasmids targeting TG2 protein and Mertk protein were constructed firstly， then the H1 promoter and RNA interfering （RNAi） sequence were cut and ligated to pAdTrack for constructing pAdTrack/TG2/Mertk. The pAdTrack/TG2/Mertk was transfected into BJ5183 bacterial cells which contained pAdEasy-1， then the plasmid was detected by enzyme digestion after recovery. Adenovirus were harvested after that pAdTrack/TG2/Mertk was infected into HEK293 cells. The virus titer was measured after repeated amplification. The RAW264.7 cells were infected by pAdTrack/TG2/Mertk， pAdTrack/TG2， pAdTrack/Mertk， and pAdTrack/green fluorescent protein （GFP）， respectively. Then the expression levels of TG2 protein and Mertk protein of mouse macrophages were detected by Western blot after infection. Results The virus titer of pAdTrack/TG2/Mertk plasmid was 6.13×1010GFU/mL. The pAdTrack/TG2/Mertk plasmid which contained 2 promoters and 2 RNAi sequences was identified successfully by enzyme digestion. Compared with pAdTrack/GFP group and pAdTrack/Mertk group （there was no significant differece between the 2 groups）， the expression levels of TG2 protein of mouse macrophages which infected with pAdTrack/TG2/Mertk or pAdTrack/TG2 decreased obviously （P＜0.01）， but there was no significant difference between the later 2 groups. Compared with pAdTrack/GFP group and pAdTrack/TG2 group （there was no significant difference between the 2 groups）， the expression levels of Mertk protein of mouse macrophages which infected with pAdTrack/TG2/Mertk or pAdTrack/Mertk decreased obviously too （P＜0.01）， but there was no significant difference between the later 2 groups. Conclusion Gene silence adenovirus vector plasmid targeting both TG2 and Mertk synchronously is constructed successfully， and the pAdTrack/TG2/Mertk can reduce the expressions of TG2 protein and Mertk protein of mouse macrophages obviously.
Objective To summarize the traits of calcium-binding tyrosine phosphorylation regulate gene and the significance in tumor incidence. Methods The domestic and foreign literatures about calcium-binding tyrosine phosphorylation regulate gene involved in the regulation of signaling pathways and research status in a variety of tumors were reviewed. Results Calcium-binding tyrosine phosphorylation regulate gene induced the abnormal proliferation of cells through multiple mechanisms. There was closely relation between the occurrence of many tumors and abnormal expression of calcium-binding tyrosine phosphorylation regulate gene. In the distribution of different epithelial tumors， the pathway of calcium-binding tyrosine phosphorylation regulate gene involved in the regulation was same， and the effect target was similar. Conclusion Further study of the calcium-binding tyrosine phosphorylation regulate gene is expected to provide a new way for clarify the occurrence and development mechanisms of tumors， and can serve as important means of early diagnosis and adjuvant therapy.
Objective To investigate the expressions of vascular endothelial growth factor-C (VEGF-C) and its receptor Flt-4 in hepatocellular carcinoma (HCC), in order to analyze the relationships among their expressions, angiogenesis, lymph-genesis, and clinicopathologic features of HCC. Methods Sixty-two cases of HCC and 15 cases of normal hepatic tissue were studied with immunohistochemical method in order to inspect the expressions of VEGF-C and Flt-4, and to calculate the microvessel density (MVD) marked by CD34 and the lymphatic vessel density (LVD) marked by Flt-4. Besides their correlations, their relations with clinicopathologic features of HCC were further analyzed. Results The positive rates of VEGF-C and Flt-4 were obviously higher in HCC than those in normal hepatic tissue (Plt;0.05, Plt;0.01). The expression of VEGF-C in HCC was remarkably related with portal vein tumor emboli, histological differentiation of HCC, and postoperative recurrence and metastasis (Plt;0.05, Plt;0.01). The expression of Flt-4 in HCC was also related with histological differentiation and postoperative recurrence (Plt;0.05, Plt;0.01). MVD was related with tumor size, TNM clinical stage, histological differentiation, portal vein tumor emboli, and postoperative recurrence and metastasis (Plt;0.05, Plt;0.01). LVD was related with histological differentiation, postoperative recurrence and metastasis (Plt;0.05, Plt;0.01). Additionally, there was positive correlation between VEGF-C and Flt-4, MVD or LVD, Flt-4 and MVD or LVD, MVD and LVD, respectively (Plt;0.01). Conclusions VEGF-C and Flt-4 are highly expressed in HCC, and are related with postoperative recurrence, are positive correlated with MVD and LVD. It suggests that VEGF-C/Flt-4 might has an effect on progression and prognosis of HCC through promoting angiogenesis and lymph-genesis.
ObjectiveTo investigate role and mechanism of protein tyrosine phosphatase 1B (PTP1B) in jejunoileal bypass to treating rats with type 2 diabetes mellitus (T2DM). MethodsTwenty-four T2DM SD rats and 24 normal SD rats were selected randomly by using random number table, then the SD rats with T2DM were randomly divided into jejunoileal bypass operation (DJBO, n=12) group and sham operation (DSO, n=12) group, the SD rats with normal food diet were randomly divided into jejunoileal bypass operation (NJBO, n=12) group and sham operation (NSO, n=12) group. Subsequently, fasting body weight (FBW), fasting plasma glucose (FPG), fasting insulin (FINS), and homeostasis model-insulin resistant (HOMA-IR) index of rats in each group were tested at different time points (before operation, on week 4 and 8 after operation). In addition, expression of PTP1B protein in skeletal muscle was determined by immunohistochemical staining and Western blot method respectively. Results① The FBW before making T2DM model had no significant difference between the rats with high-fat diet and with normal diet (P > 0.05), which on week 4 or 8 after making T2DM model in the rats with high-fat diet was significantly heavier than that in the rats with normal diet (P < 0.05). ② Before jejunoileal bypass operation, the FBW, FPG, FINS, and HOMA-IR index in the DJBO group and the DSO group were significantly higher than those in the NJBO group and the NSO group (P < 0.05), respectively, which had no significant differences between the DJBO group and the DSO group (P > 0.05) and between the NJBO group and the NSO group (P > 0.05). ③ Compared with the values before jejunoileal bypass operation, the FBW, FPG, FINS, and HOMA-IR index on week 4 or 8 after jejunoileal bypass operation were significantly decreased in the DJBO group (P < 0.05); the FBW was significantly increased on week 4 or 8 after jejunoileal bypass operation in the DSO group and the NSO group (P < 0.05), and on week 8 after jejunoileal bypass operation in the NJBO group (P < 0.05). The other indexes had no significant differences between before and after jejunoileal bypass operation in the DSO group, the NSO group, or the NJBO group (P > 0.05). ④ On week 8 after jejunoileal bypass operation, the expression of PTP1B protein in the DSO group was significantly higher than that in the DJBO group, the NSO group or the NJBO group (P < 0.05), which in the DJBO group was significantly higher than that in the NSO group (P < 0.05) or the NJBO group (P < 0.05), which had no significant difference between the NJBO group and the NSO group (P > 0.05). ConclusionJejunoileal bypass could effectively improve insulin resistance and decrease FPG level and FBW of T2DM rats through inhibiting expression of PTP1B protein in skeletal muscle of rat with T2DM.
Objective To explore the levels and the clinical significance of serum soluble Endoglin (sEng) and soluble Fms-like tyrosine kinase (sFlt-1) in patients with preeclampsia (PE). Methods Ninety-six patients with PE were included from June 2009 to June 2014. The patients were divided into mild PE group (n=54) and severe PE group (n=42), while 40 healthy pregnant women were in the control group. The general situation and laboratory testing were recorded and the serum levels of sEng and sFlt-1 were detected. All patients were routinely followed up with the recording of delivery and neonatal situation. Results The sEng and sFlt-1 levels were highest in the severe PE group [(7345.02±772.73) and (866.08±203.24) ng/L], which was followed by mild PE [(5 547.08±564.06) and (603.99±138.37) ng/L] and control group [(1 840.93±300.71) and (252.68±83.03) ng/L] (P<0.01). Levels of sEng were significantly correlated with sFlt-1 in both mild and severe PE groups. There were significantly correlations between sEng and sFlt-1 in mild or severe PE group respectively. The level of sEng and sFlt-1 was considerably positively correlated with mean arterial pressure, 24-hour urinary protein, serum creatinine, fibrinogen, umbilical artery shrink/diastole and resistance index value, but negatively correlated with prothrombin time, birth weight and the placenta weight (P<0.05). PE patients with sEng of <5 000 ng/L and sFlt-1 levels of <700 ng/L had the risk of severe complications of 6.8% and 14.0%; while patients with sEng of ≥5 000 ng/L and sFlt-1 of ≥700 ng/L had the ratio fo 40.4% and 37.0% respectively (P<0.01). Conclusion Serum levels of sEng and sFlt-1 in PE patients indicate that the severity of disease and outcomes of pregnancy.