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Objective: Using yeast surface presentation technology, secreted anti-PD-L1 single-chain antibody fragment ( sc Fv), then purify the sc Fv that specifically binds PD-L1 antigen. The sc Fv antibody gene sequence was synthesized based on the single chain antibody gene sequence. We express this sc Fv-mFc protein by using p Fuse eukaryotic expression vector to study its affinity and in vitro and in vivo inhibition of lung adenocarcinoma cells ( A549). Methods: Recombinant plasmid p Fuse-scFv was constructed by gene engineering. The recombinant plasmid p Fuse-scFv was transfected into 293 F ( human embryonic kidney cells) and cultured in serum-free Pro293 a-CDM for 72 hours, then the fusion protein was collected, and use the Rapid Protein Liquid Phase Separation and Purification System to purify the sc Fv-mFc fusion protein. Then the fusion protein and the tumor cells were detected by immunohistochemistry; the affinity of fusion protein and tumor cells was analyzed by flow cytometry; ADCC was used to determine the proliferation of tumor cells in vitro. The nude mice inoculated with lung adenocarcinoma cells, and use the fusion protein to verify its anti-tumor effect in vivo. Results: sc Fv-mFc fusion protein was secreted into serum-free culture medium by recombinant plasmid transfection into the 293 F cells; immunohistochemistry and flow cytometry showed that the fusion protein was highly expressed with the surface of PD-L1 protein;ADCC showed that the fusion protein inhibited the proliferation of tumor cells in vitro; the results of tumor-bearing mice showed that the fusion protein inhibited the growth of the tumor. At the dose of 5 mg/kg, The tumor volume growth rate decreased from 14. 90％ to3. 72％, the two independent samples t test P＜0. 05, the difference was statistically significant. Conclusion: The fusion protein containing single chain antibody was successfully prepared, which had good binding ability to A549 cells and inhibited the proliferation of tumor cells in vitro and in vivo, and provided the laboratory basis for the development of targeted anti-tumor drugs.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of postischemic treatment with endomorphin-1 (EM-1) against myocardial ischemia/reperfusion (IR) injury in rats and on extracellular signal regulated kinase 1/2 (Erk1/2)-dependent signaling pathway.</p><p><b>METHODS</b>Sprague-Dawley rats were randomly divided into 5 groups, namely the sham-operated group, IR group, EM-1 post-treatment group (EM50 group), EM-1 post-treatment group with PD98059 treatment (EM50+PD group), and PD98059 post-treatment group (PD group). The hemodynamic indexes of the rats were recorded. After reperfusion, CK-MB, LDH, CTnI, MDA, IL-6, TNF-α, and SOD activities or contents were measured, the infarct size was determined, and the expression levels of Erk1/2, P-Erk1/2 and cleaved caspase-3 were detected using Western blotting.</p><p><b>RESULTS</b>Compared with the sham group, the IR group showed significantly decreased heart rate and mean arterial pressure (P<0.05), which were increased obviously by EM-1 post-treatment (P<0.05). EM-1 post-treatment also resulted in significantly decreased LDH, CK-MB, CTnI, MDA, IL-6, and TNF-α activities or contents (P<0.05), increased SOD activity (P<0.05), reduced the infarct size (P<0.05), and increased the expression level of P-Erk protein (P<0.05). Compared with EM50 group, EM50+PD group showed significantly decreased heart rate and mean arterial pressure (P<0.05), increased LDH, CK-MB, CTnI, MDA, IL-6, and TNF-α activities or contents (P<0.05), decreased SOD activity, increased infarct size (P<0.05), and lowered expression of P-Erk protein (P<0.05).</p><p><b>CONCLUSION</b>Postischemic treatment with EM-1 protects the heart against IR injury by improving the cardiac function, inhibiting inflammation, and inhibiting oxidative stress and myocardial apoptosis, and Erk1/2 signaling pathway may be involved in this process.</p>
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the clinicopathological significance of p16(INK4A) expression and DNA ploidy status in HPV-negative uterine cervical cancers and their precursors.</p><p><b>METHODS</b>HPV-negative cervical lesions, including 20 cases of cervicitis, 20 cases of cervical intraepithelial neoplasm (CIN), 3 cases of cervical glandular intraepithelial neoplasm (CGIN), 38 cases of invasive squamous cell carcinoma (SCCs) and 15 cases of invasive adenocarcinoma were selected and subject to screening for HPV infection by PCR method. The p16(INK4A) protein expression and DNA ploidy status were studied by immunohistochemistry and flow cytometry respectively.</p><p><b>RESULTS</b>Specific expression of p16(INK4A) was seen in both the nucleus and cytoplasm of the dysplastic and malignant cells of CIN, CGIN, cervical SCC and adenocarcinoma. In contrast, no expression was present in normal and inflammatory squamous or glandular epithelium. DNA aneuploidy was significantly more frequent in invasive SCCs and adenocarcinomas than in CIN (P < 0.01). Aneuploid was also more frequent in the lymph node positive group than lymph node negative group, although no statistic significance was found. Among the 8 cases of p16(INK4A) negative SCCs, two showed DNA aneuploidy.</p><p><b>CONCLUSIONS</b>Immunohistochemical detection for p16(INK4A) can be an early diagnostic marker for HPV-negative cervical SCC and adenocarcinoma. DNA ploidy analysis may further assist the diagnosis of cervical malignancies.</p>