摘要
Objective:To investigate the affinity and toxicity of epithelial cell adhesion molecule (EpCAM) targeted nucleic acid aptamer drug conjugate SYL3C-MMAE on human gastric epithelial cells GES-1 (hereinafter referred to as GES-1 cells) and human gastric cancer cells AGS and MKN45 (hereinafter referred to as AGS cells and MKN45 cells).Methods:The experimental study was conducted. The expression level of EpCAM in gastric cancer tissues was detected using immunohistochemistry. The mRNA expression level of EpCAM in gastric cancer tissues was detected using real-time fluorescence quantitative PCR (RT-PCR). The expression level of EpCAM protein in GES-1, AGS and MKN45 cells was detected using Western blot. The affinity of SYL3C on GES-1, AGS and MKN45 cells was detected using flow cytometry. SYL3C-MMAE was synthesized through a thiol-maleimide reaction. The toxicity of drugs on GES-1, AGS and MKN45 cells was detected using CCK-8 assay. The cell cycle condition of GES-1, AGS and MKN45 cells after drug treatment was detected using propidium iodide (PI) staining. Observation indicators: (1) expression of EpCAM in gastric cancer; (2) affinity of antibodies targeting EpCAM and SYL3C on GES-1, AGS and MKN45 cells; (3) situation of drug synthesis; (4) drug toxicity and inhibition of cell cycle. Measurement data with normal distribution were represented as Mean± SD. One-way ANOVA was used for comparison among multiple groups, and pairwise comparison was conducted using the least significant difference test. Comparison of unequal variances was conducted using the Welch' t test. Measurement data with skewed distribution were represented as M(IQR), and comparison between groups was conducted using the paired rank sum test. Count data were described as absolute numbers, comparison between groups was conducted using the paired chi-square test. Results:(1) Expression of EpCAM in gastric cancer. Results of immunohistochemistry on tissue microarrays showed that the positive rate of EpCAM was 82.9%(29/35) and 22.9%(8/35) in the 35 pairs of gastric cancer and its adjacent tissues (normal tissues), respectively, showing a significant difference between them ( P<0.05). Results of RT-PCR showed that the mRNA relative expression levels of EpCAM was 1.23 (4.13) and 4.04 (1.72) in 12 pairs of gastric cancer and its adjacent tissues respectively, showing a significant difference between them ( Z=-2.67, P<0.05). Results of Western blot showed that the relative expression levels of EpCAM protein in GES-1, AGS, and MKN45 was 0, 1.00, and 0.27, respectively, with the expression level of EpCAM protein in AGS cells as the standard. (2) Affinity of antibodies targeting EpCAM and SYL3C on GES-1, AGS and MKN45 cells. Results of flow cytometry showed that antibodies targeting EpCAM and SYL3C had good affinity on AGS and MKN45 cells but no affinity on GES-1 cells. (3) Situation of drug synthesis. Results of mass spectrometry showed that the drug solution of compound formed by connecting SYL3C with monomethylorestatin E (VcMMAE) exhibited a strong peak at the molecular weight position of 16 355, consistent with the expected molecular weight of the SYL3C-MMAE complex, indicating that SYL3C-MMAE was successfully synthesized. (4) Drug toxicity and inhibition of cell cycle. Results of CCK-8 assay showed that the half maximal inhibitory concentration (IC 50) of VcMMAE on GES-1, AGS and MKN45 cells was 123.00, 30.48 and 51.83 nmol/L, respectively. The IC 50 of SYL3C-MMAE on GES-1, AGS and MKN45 cells was 241.80, 20.66 and 27.64 nmol/L, respectively. Results of PI staining and flow cytometry showed that both VcMMAE and SYL3C-MMAE could induce G2/M phase blockage in the cell cycle of GES-1, AGS and MKN45 cells. Conclusion:The SYL3C-MMAE has a good affinity on gastric cancer cells. Compared with VcMMAE, SYL3C-MMAE exhibits efficient inhibition on gastric cancer cells, but less influence on normal cells.
摘要
Objective:To identify the pathogenic mutation in a patient with Oguchi disease.Methods:A Japanese patient with Oguchi disease was enrolled in this study, and underwent a comprehensive medical history assessment and multiple ophthalmic examinations, including BCVA, OCT, color fundus photography and full field electroretinogram. Genomic deoxyribonucleic acid (DNA) was extracted from peripheral blood samples for whole exome sequencing. The gene mutation was detected, and the analysis software was used to determine the conservation of the mutation and the possible structural changes.Results:The patient, 71 years old, with consanguineous parents, complained of night blindness since early childhood. BCVA in both eyes was 0.7 and the golden-yellowish reflex appeared in the grey retina. The scotopic 0.01 ERGs showed a extinguished reaction in both eyes. The scotopic 3.0 ERGs showed a "negative" configuration with a significantly reduced a wave and a nearly absent b wave. A homozygous deletion mutation in the SAG gene (c.924delA, p.N309Tfs*12) in this patient was founded by DNA sequencing, which was predicted to generate prematurely truncated SAG protein and result in severe structural change. Homology analysis of the protein sequence indicated that the mutation resulted in an altered amino acid which was evolutionarily highly conserved among different species, strongly suggesting the potential pathogenicity of this homozygous mutation.Conclusion:The mutation c.924delA(309Tfs*12) in SAG cause Oguchi disease in this patient.
摘要
Objective To investigate the factors of families related to the outcome of rehabilitation for cerebral palsy children. Methods Families of 153 cerebral palsy children were investigated with questionnaires of family condition, knowledge of cerebral palsy, compliance, and the Eysenck Personality Questionnaire, Marital Quality Questionnaire, Social Support Scale, Symptom Check List, Happiness Index. The children were assessed with Comprehensive Assessment for Disable Children before and after rehabilitation. Results There were signifi-cant differences of compliance, knowledge of cerebral palsy, being of other healthy children, personality of extraversion/introversion and ly-ing, and mental symptom between the families of the cerebral palsy children improved or not after rehabilitation (P<0.05). Multivariate Lo-gistic regression analysis showed that, being of other healthy children, personality of extraversion/introversion and mental symptom were the independent factors. Conclusion It may relate to the outcome of rehabilitation for cerebral palsy children of family condition, personali-ty and mental health of the families, which need further intervention.