Objective To observe the effects of cobalt chloride (CoCl2)-simulated hypoxia on VEGF and TGF-β1 expression and to provide theoretical basis for deci phering the molecular mechanism of cl inical distraction osteogenesis. Methods The mandibular osteoblasts were obtained from newborn Wistar rats within 24 hours and cultured and purified through modified enzymatic digestion. The morphological and histological changes of cells were evaluated by the HE staining,the histochemical staining for ALP, the collagen I immunohistochemistry staining and the calcified nodules staining, and the growth curves were drawn. The best cells of the 3rd-passage rats were treated with CoCl2, and then immunofluorescence was used to detect the expressions of VEGF and TGF-β1 at 0, 3, 6, 9, 12 and 24 hours after culture. Results The HE staining demonstrated that the cellular forms were diverse, triangular, polygonal, circular and scaly and so on. The prominence varied in length and extended outwards. The nucleus was clearly discernible. The cytoplasma was rich and pink, with the nucleus royal purple. Sometimes 2 cell nuclei were seen. At the crowded place, cellular form was not clear, the dividing l ine was indistinct, and just the great-circle nuclear cells could be seen. The ALP immunohistochemistry staining demonstrated that the cell butcher nature appeared black pellets, the cell nucleus outl ine was unclear, and at the cell compact district, massive mascul ine cells could be seen clearly. The collagen I immunohistochemistry staining demonstrated that mascul ine cells were seen evenly, cytoplasma appeared yellowish brown especially around the nucleus. However, yellowish brown pellets were not seen in negative cells. The osteoblast calcium tubercle staining demonstrated that the cells gathered in the opaque region with the shape of tubercle after15 days of culture. After al izarin red staining, the reddish orange pigmentation appeared. At various time points, weak VEGF fluorescence was seen in the cells in the control group under the laser confocal microscope. As the hypoxia time prolonged, VEGF fluorescence of cells in the experimental group intensified, and reached the peak 9 hours after peration, and then dropped to the normal level. At various time points, TGF-β1 fluorescence was found in both groups under the laser confocal microscope, and fluorescence intensity in the control group was sl ightly ber than that in the VEGF control group. In the experimental group, TGF-β1 expression had short-term increase 3 hours after hypoxia, and reduced gradually with the prolonging of hypoxia time. Conclusion The method of culturing osteoblast from Wistar rats mandibular is practicable. The cells can be used for further studies. Moderate hypoxia can affect bone synthesis and turnover in distraction osteogenesis and up-regulate the expressions of VEGF and TGF-β1.
ObjectiveTo explore the relationship of the clinicopathological characteristics of non-small cell lung cancer (NSCLC) with the mutations of epidermal growth factor receptor (EGFR), K-ras and EML4-ALK fusion gene in cell blocks of pleural effusion (PLE). MethodsA total of 268 cytological specimens of PLE (pleural effusion), from Central Hospital of Zibo city were collected from advanced NSCLC patients between January 2012 year and June 2014 year. There were 165 male and 103 female patients at age of 53.6 (31-76) years. Qualitative diagnosis has been made in the 268 patients using PLE samples with conventional smear. Immunohistochemical staining combined with cell block section were used for further classification. There were 76 patients diagnosed as NSCLC with 39 patients of adenocarcinoma and 37 patients of squamous-cell carcinoma. In the 76 patients of lung biopsy specimens and PLE, EGFR and K-ras mutations, EML4-ALK fusions were tested. ResultsEGFR mutations rate was 34.21% (26/76). K-ras mutations rate was 6.58% (5/76). EML4-ALK fusions rate was 7.89% (6/76) at the same time. EGFR and K-ras mutations, EML4-ALK fusions were mostly found in young female adenocarcinoma patients who were non-smokers. EGFR and K-ras mutations or EML4-ALK fusions were not found in the same patient. ConclusionCytological specimens are feasible for detecting EGFR were K-ras mutations and EML4-ALK fusions. This will especially benefit to patients whose histological specimen can not be obtained.
To make a rabbit model of Perthes disease and to explore the change and its significance of VEGF expression in the femoral head. Methods Twenty-four 3-month-old New Zealand rabbits (weighing 1.6-1.8 kg) were randomly divided into experimental group (n=16) and control group (n=8). A rabbit model of Perthes disease was made by excision of left l igamentum teres and retinacular blood suppl ies of femoral head. The gross appearance, X-ray film and histological observations were made and the immunohistochemistry and VEGF mRNA in situ hybridization were carried out1, 2, 4, 8 weeks after operation. Results The rabbit model of Perthes disease was made successfully; only 1 was infected5 days after operation and was made quit. The gross appearance: The femoral heads had no necrosis changes in control group at every time. The femoral heads became coarse, tarnish and smaller, and even collapsed in experimental group. The HE staining observation: The femoral heads had no necrosis changes in control group at every time after operations. New vessels and granulation tissues grew into the necrosis part in the experimental group 4 weeks and 8 weeks after operations. New bone could be seen in repaired bone. Immunohistochemistry staining: In the epiphyseal cartilage of the femoral heads in control group, an intensive VEGF immunoreactivity (VEGF-IR) was found in the hypertrophic zone with a low level of VEGF-IR in the prol iferative zone. At 1 week after operation, the percentage of VEGF+ cells in the prol iferative zone of the femoral heads in experimental group was increased compared with that of the femoral heads in control group. The percentage of VEGF+ cells in the hypertrophic zone of the femoral heads in experimental group was significantly decreased compared with that of the femoral heads in control group. At 8 weeks after operation, VEGF-IR was observed throughout the epiphyseal cartilage surrounding the bony epiphysis in the femoral heads in experimental group. The percentage of VEGF-positive cells in the prol iferative zone of the femoral heads in experimental group was significantly increased compared with that of the normal heads. The hypertrophiczone of the femoral heads in experimental group had a similar percentage of the VEGF+ cells to the femoral heads in control group when endochondral ossification was restored at 8 weeks. There were statistically significant differences in the ratios of VEGF+ cells in the prol iferative zone of femoral head 1, 2, 4, 8 weeks after operations (P lt; 0.01); in the ratios of VEGF+ cells in the hypotrophic zone of femoral head 1, 2, 4 weeks after operations (P lt; 0.01) between experimental group and control group. In situ hybridization results: The results were similar to that of histology. VEGF mRNA expression in the hypertrophic zone of epiphyseal catilage after necrosis were lower. VEGF mRNA expression in the prol iferative zone of epiphyseal catilage after necrosis increased. VEGF mRNA expression in the hypertrophic zone of epiphyseal cartilage in experimental group could be seen again after endochondral ossification was repaired. Conclusion It is possible that VEGF may act as a key regulator that couples angiogenesis, cartilage remodel ing, and ossification after ischemic damage to restore endochondral ossification in the epiphyseal cartilage.
Objective To compare the long-term cost-utility of three first-generation EGFR-TKIs targeted drugs, gefitinib, icotinib, and erlotinib as first-line treatments for advanced non-small cell lung cancer (NSCLC). Methods Real-world data were collected from 1 511 patients with advanced NSCLC treated with first-generation EGFR-TKIs as first-line treatment at West China Hospital of Sichuan University from 2009 to 2019. A three-state Markov model was established to evaluate the clinical efficacy, safety and cost-utility of three first-generation EGFR-TKIs targeted drugs. The transition probability of each state was obtained by survival analysis, the direct and indirect costs were calculated by the bottom-up method, the health utility value was obtained through literature research, the incremental cost effectiveness ratio (ICER) and quality-adjusted life years (QALYs) were calculated, and sensitivity analyses and Monte Carlo simulations were performed. Results There was no significant difference in clinical efficacy among the three first-generation EGFR-TKIs in the treatment of NSCLC. The incidence of skin rash and liver injury caused by gefitinib was significantly higher than that caused by icotinib and erlotinib (P<0.05). The average economic burden of patients treated with icotinib was the lowest (CNY 192 535.3) (P<0.01). The cost-utility ratio of icotinib (CNY 132 985.9/QALYs) was much lower than that of gefitinib (CNY 205 005.3/QALYs) and erlotinib (CNY 172 893.1/QALYs). Conclusion Compared with the three first-generation EGFR-TKIs drugs, icotinib is the most cost-effective.
Objective To construct a green fluorescent protein expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28, containing anti-TAG72 single chain variable fragment (scFv) fused into the transmembrane and intracellular domain of the signal-transducing chain of CD28 gene, and to transfect it into peripheral blood mononuclear cells． Methods Recombinant transmembrane and intracellular domain of CD28 cDNA and anti-TAG72 scFv cDNA fragment was subcloned into pEGFP-C3 vector. Recombinant clones were selected by Kanamyein， and then identified by PCR， enzyme digestion analysis and DNA sequencing. The recombinant plasmid was transfected into peripheral blood mononuclear cells by means of lipofection. The recombinant protein expression was confirmed by immunocytochemistry, laser scanning confocal microscope, PCR and Western blot analysis. Results The fused gene fragment of anti-TAG72 scFv-CD28 was successfully inserted into pEGFP-C3 plasmid, and it was confirmed by enzyme digestion and DNA sequencing. The fused anti-TAG72 scFv-CD28 gene and its protein was identified in peripheral blood mononuclear cells． Conclusion The eukaryotic expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28 was successfully constructed and transiently expressed in peripheral blood mononuclear cells， which would lay a foundation for further studies on the role of it to activate tumor-associated antigen-specific T lymphocyte, for generating of modified T lymphocytes targeting gastrointestinal tumors.
ObjectiveTo detect the expressions of signal peptide-CUB-EGF-like domain containing protein 3 (SCUBE3) and specificity protein 1 (SP1) in breast cancer tissues, and explore relations between their protein expressions and clinicopathologic features or prognosis.MethodsFrom February 2013 to October 2015, the breast cancer tissues and the corresponding adjacent normal breast tissues of 80 women patients with breast cancer in the Mianyang Central Hospital were selected, and the expressions of SCUBE3 and SP1 proteins in the tissues were detected by immunohistochemistry. The relations between the expressions of SCUBE3 and SP1 and clinicopathologic parameters of breast cancer were analyzed, the correlation between the SCUBE3 and SP1 was analyzed by Spearman rank correlation analysis. Kaplan-Meier method was used to analyze the survival of patients with breast cancer; and Cox proportional hazards regression model was used to analyze the risk factors of overall survival of patients with breast cancer.ResultsThe positive rates of SCUBE3 and SP1 proteins expressions in the breast cancer tissues were higher than those in the corresponding adjacent normal breast tissues (P<0.05). The positive rates of SCUBE3 and SP1 protein expressions were higher in the breast cancer tissues with lymph node metastasis and molecular subtypes of Luminal A or B (P<0.05), and the positive rates of SCUBE3 protein expression were higher in the breast cancer tissues with TNM stage Ⅱ–Ⅳ and high Ki67 (P<0.05). The retsult of Spearman rank correlation analysis showed that the positive rates of SCUBE3 and SP1 proteins expressions in the breast cancer tissues was positive correlation (χ2=7.979, rs=0.316, P=0.005). Kaplan-Meier curve showed that the overall survival of the patients with positive expression of SCUBE3 or SP1 protein was worse than that of the patients with negative expression (χ2=4.042, P=0.044; χ2=10.676, P=0.001). The Cox proportional hazards regression model multivariate analysis showed that the positive SCUBE3 (HR=6.020, P=0.016), positive SP1 (HR=4.077, P=0.018), lymph node metastasis (HR=3.518, P=0.017), and higher Ki67 expression (HR=7.989, P<0.001) were the independent risk factors of overall survival for the patients with breast cancer.ConclusionPositive rates of SCUBE3 and SP1 proteins expressions in breast cancer tissues are higher and there is a positive correlation between them, which are closely related to clinicopathologic parameters such as lymph node metastasis and molecular subtypes and prognosis of patients with breast cancer.
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.
The 2025 American Society of Clinical Oncology (ASCO) meeting was held in Chicago. At the annual conference, research on the treatment of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) once again took the spotlight. Combination therapy strategies have demonstrated the potential to overcome resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) and prolong survival. Meanwhile, progress has also been made in individualized treatment strategies for young patients and those with fibrotic interstitial lung disease. However, the complexity of resistance mechanisms, special treatment considerations for different populations, and the impact of socioeconomic factors on treatment accessibility remain challenges in the field of EGFR-mutant NSCLC treatment. In the future, it is necessary to further explore more effective treatment regimens and expand the accessibility of precision medicine to maximize patient benefits.
ObjectiveTo reveal the true value of plasma detection of epidermal growth factor receptor (EGFR) mutation for early-stage non-small cell lung cancer (NSCLC) gene diagnosis and to predict survival prognosis. MethodsTissue samples of positive EGFR mutations by using amplification refractory mutation system (ARMS) method were surgically resected from 198 patients with stage I-IV NSCLC between February 2014 and June 2015 in Tangdu hospital. Paired blood samples were collected before surgery. And the cellfree DNA (cfDNA) in plasma was extracted, plasma EGFR mutations were detected by real-time polymerase chain reaction (PCR). Concentration of cfDNA was measured by ultraviolet spectrophotometry. Follow-up observation for stage ⅢA patients was put into force after surgery. Kaplan-Meire was used in survival analysis. ResultsThe sensitivity of EGFR mutation for the 198 paired tissues and plasma samples was 17.2%.The sensitivity was positively correlated with TNM stage and negatively correlated with tumor differentiation. The sensitivity of sage ⅢA was 33.3%, significantly higher than that of the patients at stage ⅠA (1.6%, P=0.000) and stage ⅠB (7.9%, P=0.004). The sensitivity of poor differentiation was 36.8%, significantly higher than that of high differentiation (0.0%, P=0.000) and moderate differentiation (15.7%, P=0.010). There was no correlation between plasma cfDNA concentration and patient characteristics. Survival analysis showed that plasma detection was a vital factor for predicting postoperative survival prognosis of stage ⅢA patients (P=0.014). ConclusionTissue samples cannot be replaced by plasma samples for epidermal growth factor receptor (EGFR) mutation test in early-stage NSCLC patients, currently. When the sensitivity increases dramatically in the plasma samples of stage ⅢA NSCLC and poor differentiation tumor, we recommend using plasma detection for gene diagnosis, dynamic monitoring of EGFR mutations in stage ⅢA or poorly differentiated tumors, especially in NSCLC patients whose tissue samples cannot be obtained by surgery. And plasma EGFR detection is a valuable method of forecasting survival prognosis for locally advanced NSCLC patients.
ObjectiveTo investigate the expression of tumor metastasis associated genes-1 (MTA1) and vascular endothelial growth factor-C (VEGF-C) in esophageal squamous cell carcinoma (ESCC) and the relationship between them and lymphangiogenesis. MethodA total of 107 patients who received excision for ESCC in the Cardiothoracic Surgery Department of Suining Central Hospital from March 2013 through January 2014 were enrolled. And the paraffinembedded esophageal tissues in 56 healthy persons were collected. The expression of MTA1 and VEGF-C in ESCC was detected using the immunohistochemical method. And D2-40 was used to label the micro-lymphatic endothelial cells of the tumor tissues while the micro-lymphatic vessel density (LVD) was counted. Meanwhile, a statistical analysis was performed for the relationship between MTA1 with VEGF-C and clinical pathological parameters. ResultsThe expression rates of MTA1 protein and VEGF-C protein in ESCC (50.4% and 58.8%, respectively) were higher than those in normal esophageal tissues with a statistical difference (P<0.05). Besides, their high expression rates in stage T3/T4 ESCC and lymph node metastasis group were significantly higher than those in stage T1/T2 ESCC and metastasisfree group, with statistical differences (P<0.05). The high expression rates of MTA1 and VEGF-C protein in ESCC with different TNM stages were compared using Kruskal-Wallis test with statistical differences (P<0.05). Moreover, a positive correlation existed in the expression level between MTA1 protein and VEGF-C protein of ESCC (Spearman coefficient r=0.512, P=0.000). And LVD of the high expression group for MTA1 protein and VEGF-C protein was statistically different from that of the low expression group (P<0.05). ConclusionThe expression of MTA1 is positively correlated with the expression of VEGF-C in ESCC. And they may co-promote lymphangiogenesis and lymphatic metastasis in ESCC. Therefore, both can be used as the laboratory indicators to determine the prognosis of ESCC.