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Objective To construct an objective analysis system of corneal nerve tortuosity and detect the changes of corneal subbasal nerve tortuosity in patients with dry eye and diabetes. Methods GradeⅠtoⅣnerve tortuosity were evaluated and 80 photos of each grade were randomly chosen from the in vivo confocal microscopy library. Nerve fibers were extracted,segmented and then analyzed by 6 tortuosity related parameters including L C, Seg L C mean,Cur mean,Specific p,ICM and SCC mean. After verifying the validaty of parameters above,a cross-sectional study was conducted. Subjects were collected from June,2018 to February,2019 in Peking University Third Hospital,and were divided into healthy control group (28 persons 56 eyes),dry eye without diabetes group (28 patients 56 eyes),diabetes without dry eye group(24 patients 48 eyes),diabetes with dry eye group (23 patients 46 eyes) . Basic and dry eye information includes sex,age,ocular surface disease index ( OSDI) ,tear film break-up time (TBUT),Schirmer Ⅰ test (SⅠt) and corneal fluorescence staining (CFS) score. Fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) were detected in diabetic patients. Cochet-Bonnet examination (C-BE) was detected to evaluate corneal sensation and 2 corneal subbasal nerve photos of each eye were selected for effective tortuosity and density related parameters analysis. Data was analyzed by SPSS and diagnostic test were perfomed by MedCalc. This study followed the Declaration of Helsinki. This study protocol was approved by Ethic Committee of Peking University Third Hospital ( No. IRB00006761-M2017354 ) . Written informed consent was obtained from each subject prior to entering study cohort. Results L C,Seg L C mean,Cur mean,Specific p,ICM and SCC mean increased as the nerve tortuosity increased from Grade Ⅰ to Grade Ⅳ,with an overall significance among 4 groups (F=39. 100, 36. 367,57. 743,4. 043,6. 818,33. 493;all at P<0. 01). Among the above 6 parameters,Cur mean and L C of any two groups were of significant difference (all at P<0. 01). Twenty three to twenty eight persons were enrolled in each group of the cross-sectional study. Sex and age were comparable among 4 groups. Diagnostic criteria were met in dry eye and diabetes. Corneal sensation parameter C-BE decreased in diabetes without dry eye group and diabetes with dry eye group compared with healthy control group ( all at Adj P<0. 05 ) , other than in dry eye without diabetes group (AdjP≥0. 05). Nerve density of diabetes without dry eye group and diabetes with dry eye group was lower compared with healthy control group(all at P<0. 001),while no significant difference between dry eye without diabetes group and healthy control group(P≥0. 05). Among the effective parameters of tortuosity,L C,Cur mean,Seg L C mean and SCC mean of dry eye without diabetes group,diabetes without dry eye group,diabetes with dry eye group were higher compared with healthy control group ( all at P<0. 05 ) . Diagnostic tests of tortuosity related parameters all showed an area under curve (AUC) from 0. 5 to 0. 7. Conclusions L C and Cur mean can be used to analyze corneal nerve curvature more reliably. Compared with normal volunteers,patients of dry eye or diabetes show higher corneal subbasal nerve tortuosity.
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Objective To investigate the effect of signal transducer and activator of transcription-3(STAT-3)-mediated non-dependent vascular endothelial growth factor receptor-2(VEGFR-2) pathway on the radiosensitivity of non-small cell lung cancer (NSCLC) cells and its possible mechanism.Methods NSCLC cells were divided into control group, apatinib (VEGFR-2 inhibitor) group, apatinib+S3I-201(STAT-3 inhibitor) group, radiation (RT) group, RT+apatinib group, and RT+apatinib+S3I-201 group.Q-PCR and Western blot were used to measure the mRNA and protein expression of VEGFR-2, STAT-3, other proteins involved in the signaling pathway, hypoxia-inducible factor 1α(HIF-1α), and cyclin D1.The cellular radiosensitivity was determined by colony-forming assay and cell apoptosis and cell cycle distribution were evaluated by flow cytometry.Results For Calu-1 cells, there was no significant difference in the expression of VEGFR-2 mRNA and STAT-3 mRNA between the apatinib group and the control group (P>0.05);the apatinib group had significantly lower expression of HIF-1α mRNA and cyclin D1 mRNA than the control group (P<0.05);the apatinib+S3I-201 group had significantly lower mRNA and phosphorylated protein expression of VEGFR-2, STAT-3, and related downstream target proteins than the control group (F=304.54, P<0.01;F=118.99, P<0.01, F=144.34, P<0.01;F=529.66, P<0.01);compared with the control group, the apatinib+S3I-201 group and the apatinib group showed increases in apoptosis rate and proportion of cells in G2+M phase, and the apatinib+S3I-201 group had significantly greater increases than the apatinib group (F=72.37, P<0.01).Compared with Calu-1 cells, the radiosensitizing effect of apatinib on A549 cells was limited (SER[sensitizer enhancement ratio]=1.39).The radiosensitizing effect of apatinib+S3I-201 on A549 cells was significantly higher than that of apatinib (SER:1.72 vs.1.39, P=0.000).Conclusions The inhibition of STAT-3 can enhance the radiosensitivity of NSCLC cells.When VEGFR-2 is inhibited by apatinib, activated STAT-3 regulates the expression of cyclin D1 directly or indirectly to affect the radiosensitivity of NSCLC cells.The inhibition of VEGFR-2 and STAT-3 has a good radiosensitizing effect on NSCLC cells.
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Objective@#To explore radiosensitivity-associated genes in esophageal squamous cell carcinoma by targeted sequencing panel.@*Methods@#The peripheral blood from 22 esophageal squamous cell carcinoma (ESCC) patients received radiotherapy alone were collected, respectively. The genomic DNA (gDNA) of peripheral blood was extracted and used to create a library of gDNA restriction fragments. The gDNA restriction fragments were hybridized to the HaloPlex probe capture library, which comprises 356 cancer genes selected from the Catalogue of Somatic Mutations in Cancer (Cosmic) database of 2011 updated edition. The sequencing data were aligned by the Genome Analysis Toolkit GATK (version 3.0) and Picar. The single nucleotide polymorphism and inserted-deletion (SNP/InDel) variations were annotated by online database. The pathway enrichment was analyzed by Ingenuity Pathway analysis (IPA). Moreover, according to the short-period curative effect, 22 patients were divided into two groups: the radiation- sensitivity group (CR+ PR) and the radiation-resistant group (PD+ SD). The nonsynonymous mutation sites were statistically analyzed and the genes associated with radiosensitivity of ESCC were screened.@*Results@#More than 97% sequencing reads were aligned to human genome reference sequence and more than 90% sequencing reads were the target sequences. SNP/InDel database annotation results showed that the mutations of 22 cases mainly distributed in exons, and the mutant types were mainly missense and synonymous single nucleotide variant (SNV). There were 23 genes of high-frequency mutation associated with esophageal cancer. Pathway enrichment by IPA showed that 3 pathways were associated with the development of esophageal cancer, which were roles of BRCA1 in DNA damage response pathway, DNA double-strand break repair by non-homologous end joining pathway and ATM signaling pathway. According to the curative effect, five genes including mismatch repair system component (PMS1), fibronectin 1(FN1), mutL homolog 1 (MLH1), B-Raf proto-oncogene, serine/threonine kinase (BRAF), patched 1 (PTCH1) and cytochrome P450 family 2 subfamily C member 19 (CYP2C19) were associated with radiosensitivity of ESCC patients.Moreover, the PTCH1 was mutated in all of 22 ESCC patients, while the variations of rs199476092 and rs202111971 sites of PTCH1 were only identified in the radiation-resistant group.@*Conclusions@#We find that the variations of rs199476092 and rs202111971 in the encoding region of PTCH1 gene are significantly associated with radiosensitivity of ESCC patients.
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Objective To investigate the effects of vascular endothelial growth factor receptor?2 ( VEGFR?2) on proliferation, migration, invasion, and apoptosis after radiotherapy in lung cancer cell line Calu?1, and to explore the probable mechanisms. Methods Small interference RNA ( siRNA )?mediated silencing of VEGFR?2 gene was performed on Calu?1 cells, and the mRNA and protein expression of VEGFR?2 was determined by quantitative real?time PCR and Western blot, respectively. The cells were divided into control group, vascular endothelial growth factor ( VEGF ) group, VEGFR?2 specific siRNA (siKDR) group, and siKDR+VEGF group. The changes in proliferation, migration, and invasion were evaluated by the CCK8 assay, cell scratch wound?healing assay, and transwell migration assay, respectively. The protein expression of VEGFR?2 and proteins in the related downstream signaling pathway was measured by Western blot. Apoptosis in each group was determined after radiotherapy. Results After RNA interference?mediated silencing of VEGFR?2, the mRNA and protein expression of VEGFR?2 was significantly reduced ( P=0. 001,P=0. 000);the proliferation, migration, and invasion of Calu?1 cells were also significantly reduced ( P=0. 000,P=0. 000,P=0. 000);the phosphorylation levels of AKT, ERK 1/2, and p38 were significantly reduced in Calu?1 cells ( P=0. 336,P=0. 986,P=0. 553);the apoptosis in Calu?1 cells was significantly elevated ( P=0. 0012);the protein expression of HIF?1α was significantly inhibited ( P= 0. 016 ) . Conclusions The VEGFR?2 gene silencing significantly inhibits several physiological functions of Calu?1 cells and elevates the apoptosis rate after radiotherapy.
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Objective To investigate the radiosensitivity of stem cells in pancreatic adenocarcinoma PANC‐1 cell line and the possible mechanism of its radiation resistance .Methods Using flow cytometry ,the cells were isolated and sorted into CD44+CD24+ ,CD44-CD24+ ,CD44+CD24- ,and CD44-CD24- .Multi‐target click model was used to fit cell survival curves and deter‐mine the sensitizer enhancement ratio .The apoptosis and cycle distribution of the four cell subsets were determined using flow cy‐tometry ,and the level of ROS was determined by DCFH‐DA probe .Results The ratio of CD44+ and CD24+ in the sorted PANC‐1 cell line was 92 .0% and 4 .7% respectively .Before radiation ,there was no statistically significant difference between four groups (P>0 .05);After treated with 6MV‐X ray ,The ratio of apoptosis was the lowest in CD44+CD24+ (P<0 .01);The percentage G0/G1 cell was the highest in CD44+CD24+ (P<0 .01) ,the sensitizer enhancement ratio of CD44+ CD24- ,CD44-CD24+ and CD44-CD24- were 1 .61 ,1 .81 ,1 .94 ,respectively .The level of ROS in CD44+CD24+ was lower (P<0 .01) .Conclusion Tumor stem cells of pancreatic adenocarcinoma have properties of a lower level of ROS and relative stationary that maybe the reasons of radio resist‐ance .
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Objective To determine the effects of endostatin on the expression of vascular endothelial growth factor receptor?2 ( VEGFR?2 ) in non?small cell lung cancer cells ( human A549 lung adenocarcinoma cells and human Calu?1 lung carcinoma cells) , and to investigate the possible mechanisms underlying its radiosensitizing effect. Methods The CCK8 method was used to determine the inhibitory effect of endostatin on cell proliferation and calculate the drug concentration that caused a 20% reduction in cell proliferation within 24 h ( IC20 ) . RT?PCR and Western blot assays were used to assess the mRNA and protein expression of VEGFR?2, proteins within its related signaling pathways, and HIF?1α, respectively. The radiosensitivity of cells in each group was determined by colony formation assay;cell apoptosis and cell cycle distribution were determined by flow cytometry. Comparison of mean values between multiple samples was made by one?way analysis of variance, and comparison of mean values between two samples was made by t test. Results Endostatin significantly inhibited the proliferation of Calu?1 cells ( F=50?36,P<0?01) with an IC20 of 296?5 μg/ml;the mRNA and protein expression of VEGFR?2 and HIF?1α was also significantly inhibited in endostatin?treated Calu?1 cells ( F=25?43,10?44, all P<0?05) . Moreover, the phosphorylation of Akt, ERK 1/2, and p38 was significantly reduced in endostatin?treated Calu?1 cells ( F=2?89,0?24, 1?09, all P<0?05) . The radiosensitivity enhancement ratios for Calu?1 cells and A549 cells were 1?38 and 1?09, respectively. Endostatin significantly induced apoptosis ( F=44?15, P<0?01) and G2/M blockage ( F= 104?24, P< 0?01 ) in Calu?1 cells. Conclusions Endostatin induces apoptosis and enhances radiosensitivity in Calu?1 cells with high expression of VEGFR?2, but it has a limited impact on A549 cells with low expression of VEGFR?2.
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Refractoriness of acute myeloid leukemia (AML) cells to chemotherapeutics represents a major clinical barrier. Suicide gene therapy for cancer has been attractive but with limited clinical efficacy. In this study, we investigated the potential application of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) based system to inhibit chemoresistant AML cells. We first generated Ara-C resistant K562 cells and doxorubicin-resistant THP-1 cells. We found that the HSV-TK/GCV anticancer system suppressed drug resistant leukemic cells in culture. Chemoresistant AML cell lines displayed similar sensitivity to HSV-TK/GCV. Moreover, HSV-TK/GCV killing of leukemic cells was augmented to a mild but significant extent by all-trans retinoic acid (ATRA) with concomitant upregulation of Connexin 43, a major component of gap junctions. Interestingly, HSV-TK/GCV killing was enhanced by expression of vesicular stomatitis virus G glycoprotein (VSV-G), a fusogenic membrane protein, which also increased leukemic cell fusion. Co-culture resistant cells expressing HSV-TK and cells stably transduced with VSV-G showed that expression of VSV-G could promote the bystander killing effect of HSV-TK/GCV. Furthermore, combination of HSV-TK/GCV with VSV-G plus ATRA produced more pronounced antileukemia effect. These results suggest that the HSV-TK/GCV system in combination with fusogenic membrane proteins and/or ATRA could provide a strategy to mitigate the chemoresistance of AML.