Objective To investigate the effect of co-stimulatory molecular CD137 and CD28 on the cell proliferation, IL-2 secretion and cell apoptotic rate of activated T cells in naturally senile mice and subacute senile mice induced by D-galactose. Methods Seven-week-old BALB/c male mice were divided into D-galactose induced subacute senile group (D-gal group) , control group and young group randomly. Subacute senile mice model was established by back hypodermic injection of D-galactose (120 mg/kg, dissolved in 0. 1 ml distilled water) everyday for five month. Control group was established by injection of 0. 1 ml distilled water everyday for five month. Young group was injected with nothing. And 16-month-old BALB/c male mice was taken as aged group. The spleen T cells of each group were isolated and activated in vitro stimulation with ConA + IgG, ConA + CD137mAb or ConA + CD28mAb. The cell proliferation, apoptotic rate and IL-2 concentration in cell culture supernate of T cells were detected. Results (1) The cell proliferation (0.422?0.057, A), IL-2 secretion(0.632?0.066, A)and apoptotic rate(68.0%?2. 4%) of T cells in D-gal group stimulated in vitro with ConA+IgG showed no significant difference when compared with those of aged group. Compared with young and control group, activation of T cell in D-gal and aged groups were significantly decreased; (2) Cell proliferation, IL-2 secretion and cell survival of T cells in D-gal group and aged group were significantly promoted by both ConA + CD137mAb [(0. 639?0. 053, A) , (1.119?0.035,A), (53.3%?2.4)%, respectively] and ConA +CD28mAb. CD137 mAb had less effect on both groups than did CD28 mAb. Conclusions (1) Similar age-associated alterations happen in T cells of both D-gal group and aged group. (2) CD137 and CD28 can promote the activation and survival of T cells in aged and D-gal group. But CD28 has stronger effect on regulation of T cells than CD137.

BACKGROUNDTo investigate the expressions of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) and their correlation with metastasis and prognosis in human lung cancer.

METHODSImmunohistochemical S-P method was used to detect the expression of MMP-9 and TIMP-1 in 65 lung cancer tissues, 35 hyperplastic and dysplastic epithelium from patients with non-cancerous pulmonary diseases, and 30 normal epithelial tissues of the lung.

RESULTSThe positive expression rates of MMP-9 in normal tissue, hyperplastic or dysplastic epithelium, and lung cancer tissue were 16.7%(5/30), 42.9%(15/35) and 72.3%(47/65) respectively, whereas the positive rates of TIMP-1 expression in normal tissue, hyperplastic or dysplastic epithelium, and lung cancer tissue were 6.7%(2/30), 28.6%(10/35) and 50.8%(33/65) respectively. Significant differences of the expression rates of MMP-9 and TIMP-1 were found between lung cancer and normal groups, between lung cancer and hyperplasia groups, and between hyperplasia and normal groups (P < 0.05). Small cell carcinoma and adenocarcinoma had higher MMP-9 expression than squamous cell carcinoma (P < 0.025). Expression rate of MMP-9 had negative relation with cell differentiation of lung cancer (P < 0.05), and positive relation with TNM stage (P < 0.025). Between the survival time < 2 years group and ≥2 years groups, both the expressions of MMP-9 and TIMP-1 had significant difference (P < 0.05 ). The expression of MMP-9 was closely related to metastasis of lung cancer (P < 0.005), but the expression of TIMP-1 was not related to metastasis.

CONCLUSIONSOverexpression of MMP-9 may appear in precancerous lesion and at the early stage of lung cancer. Activation of MMP-9 gene may be an important factor for oncogenesis of the lung. MMP-9 and TIMP-1 may play important roles in lung cancer invasion and metastasis, their overexpression could act as a reference to evaluate metastasis and unfavourable prognosis of lung cancer.

BACKGROUNDTo study the specific expression of tumor-related genes (p53, bcl-2 and c-myc) in non small cell lung cancer with neuroendocrine differentiation (NSCLC-NE).

METHODSThe expression of neuron-specific enolase (NSE), chromogranin A (CgA), synaptophysin(Syn), c-myc, bcl-2 and p53 was detected in 60 surgically resected and paraffin-embedded non-small cell lung cancer (NSCLC) specimens by immunohistochemistry (S-P method).

RESULTSThe positive rates of NSE, CgA, Syn expressed in 60 cases of NSCLC were 45.00%(27/60), 13.33%(8/60), 31.67% (19/60) respectively. According to the results of these three markers, 41.67%(25/60) of 60 specimens was proved to be as NE differentiation cancer. The NE differentiation in NSCLC was remarkably related to differentiation of tumor cells (P < 0.05). NSCLC-NE had a higher metastatic rate (P < 0.05) and a higher clinical staging (P < 0.05) than NSCLC without NE differentiation. The positive rates of bcl-2, p53 and c-myc expression in NSCLC-NE were 68.00% (17/25), 80.00% (20/25), 68.00% (17/25) respectively, and the expression of bcl-2 and p53 was closely related to NE differentiation (P < 0.05).

CONCLUSIONSA certain part of NSCLC have NE differentiation, which has different biological features from NSCLC without NE differentiation. High expression of bcl-2 and mutant p53 can be observed in NSCLC-NE, and bcl-2/Bax unbalance associated with p53 mutation may play an important role in oncogenesis and development of NSCLC-NE.

BACKGROUNDTo study the expression of survivin mRNA in non-small cell lung cancer (NSCLC), and to explore its relationship with carcinogenesis, development invasion and metastasis of NSCLC.

METHODSIn situ hybridization was applied to detect survivin mRNA expression in 12 normal bronchial epithelium, 9 dysplasia, 34 NSCLC and 12 metastatic lymph nodes. The relationship between survivin expression and clinicopathological characteristics was analyzed.

RESULTSIn normal bronchial epithelium, dysplasia, NSCLC and metastatic lymph nodes, the positive rate of survivin mRNA expression were 16.67% (2/12), 33.33% (3/9), 61.76% (21/34), and 91.67% (11/12), respectively. There were significant differences in survivin mRNA expression between lung cancer and normal bronchial epithelium ( P < 0.01), as well as between metastatic lymph nodes and normal bronchial epithelium ( P < 0.001). There were remarkably higher survivin mRNA expressions in poor- and moderate-differentiated groups than that in well-differentiated group ( P =0.003, P =0.004). The expression of survivin mRNA was not related to histologic classification and lymph node status ( P > 0.05, P > 0.05).

CONCLUSIONSSurvivin mRNA expression may play an important role in the carcinogenesis and development of NSCLC. It may be a new target in gene therapy of lung cancer through blocking or down-regulating survivin mRNA expression to recover the normal regulation mechanism of apoptosis.

Objective To investigate whether modification with IL-21 and CCL19 enhances killing and tumor-infiltrating efficiency of NKP30 CAR-T cells in lung cancer.Methods The modified IL-21-CCL19 NKP30 CAR-T cells expressing IL-21 and CCL19 fusion gene was constructed based on NKP30 CAR-T cells and stimulated with CD3CD28 antibodies and IL-2.The immunophenotype and migration of the cells in the presence of IL-21 were investigated using flow cytometry and migration experiments.Lactate dehydrogenase(LDH)release and sphere formation assays were used to assess the killing and infiltration capabilities of CAR-T cells,and the secretion levels of IFN-γ,IL-21 and CCL19 were determined with enzyme-linked immunospot assay(ELISPOT)and ELISA.A zebrafish model bearing HCG-27 cell xenograft was established by microinjection of the tumor cells into the yolk sac followed 24 h later by injection of the immune cells at the same site,and the fluorescence signals were captured using a fluorescent microscopy.Results The NKP30 ligand B7H6,which was almost undetectable in normal tissues and blood cells,was highly expressed(over 90%)in lung cancer cells.Compared with NKP30 CAR-T cells and conventional T cells,IL-21-CCL19 NKP30 CAR-T cells exhibited stronger proliferative and migration capabilities with the formation of central memory T cells.The reduced expressions of CTLA4 and PD1 in the constructed cells resulted in enhanced killing efficiency against lung cancer cells accompanied by significantly increased production of IFN-γ,IL-21 and CCL19.In the zebrafish models,CAR-T cells exhibited stronger cytotoxicity and proliferative abilities than typical T cells,but these differences were not statistically significant between the two CAR-T cells.Conclusion Modification of NKP30 CAR-T cells with IL-21 and CCL19 facilitates their access into solid tumors for more effective tumor cell killing while producing a large number of memory T cells.

Objective To investigate whether modification with IL-21 and CCL19 enhances killing and tumor-infiltrating efficiency of NKP30 CAR-T cells in lung cancer.Methods The modified IL-21-CCL19 NKP30 CAR-T cells expressing IL-21 and CCL19 fusion gene was constructed based on NKP30 CAR-T cells and stimulated with CD3CD28 antibodies and IL-2.The immunophenotype and migration of the cells in the presence of IL-21 were investigated using flow cytometry and migration experiments.Lactate dehydrogenase(LDH)release and sphere formation assays were used to assess the killing and infiltration capabilities of CAR-T cells,and the secretion levels of IFN-γ,IL-21 and CCL19 were determined with enzyme-linked immunospot assay(ELISPOT)and ELISA.A zebrafish model bearing HCG-27 cell xenograft was established by microinjection of the tumor cells into the yolk sac followed 24 h later by injection of the immune cells at the same site,and the fluorescence signals were captured using a fluorescent microscopy.Results The NKP30 ligand B7H6,which was almost undetectable in normal tissues and blood cells,was highly expressed(over 90%)in lung cancer cells.Compared with NKP30 CAR-T cells and conventional T cells,IL-21-CCL19 NKP30 CAR-T cells exhibited stronger proliferative and migration capabilities with the formation of central memory T cells.The reduced expressions of CTLA4 and PD1 in the constructed cells resulted in enhanced killing efficiency against lung cancer cells accompanied by significantly increased production of IFN-γ,IL-21 and CCL19.In the zebrafish models,CAR-T cells exhibited stronger cytotoxicity and proliferative abilities than typical T cells,but these differences were not statistically significant between the two CAR-T cells.Conclusion Modification of NKP30 CAR-T cells with IL-21 and CCL19 facilitates their access into solid tumors for more effective tumor cell killing while producing a large number of memory T cells.