Necroptosis plays a crucial role in the exacerbation of retinal injury after blunt ocular trauma.

Retinal injury after blunt ocular trauma may directly affect prognosis and lead to vision loss. To investigate the pathological changes and molecular mechanisms involved in retinal injury after blunt ocular trauma, we established a weight drop injury model of blunt ocular trauma in male Beagle dogs. Hematoxylin-eosin staining, immunofluorescence staining, western blotting, and TUNEL assays were performed to investigate retinal injury within 14 days after blunt ocular trauma. Compared with the control group, the thicknesses of the inner and outer nuclear layers, as well as the number of retinal ganglion cells, gradually decreased within 14 days after injury. The number of bipolar cells in the inner nuclear layer began to decrease 1 day after injury, while the numbers of cholinergic and amacrine cells in the inner nuclear layer did not decrease until 7 days after injury. Moreover, retinal cell necroptosis increased with time after injury; it progressed from the ganglion cell layer to the outer nuclear layer. Visual electrophysiological findings indicated that visual impairment began on the first day after injury and worsened over time. Additionally, blunt ocular trauma induced nerve regeneration and Müller glial hyperplasia; it also resulted in the recruitment of microglia to the retina and polarization of those microglia to the M1 phenotype. These findings suggest that necroptosis plays an important role in exacerbating retinal injury after blunt ocular trauma via gliosis and neuroinflammation. Such a role has important implications for the development of therapeutic strategies.

Frequency cumulative effect of subthreshold energy laser-activated remote phosphors irradiation on visual function in guinea pigs.

AIM: To explore the effects of laser-activated remote phosphors (LARP) on visual function in guinea pigs. METHODS: Electroretinogram (ERG) of guinea pigs were observed after LARP irradiation at different frequencies and irradiation times. We evaluated the expression of rhodopsin, ß-catenin, connexin36, calretinin, and calbindin in the retina of guinea pigs and measured the density of photoreceptor cells after high-frequency LARP irradiation. RESULTS: After LARP irradiation, the ERG results showed that the amplitude of the dark-adapted 3.0 b-wave of the model eye was lower than that of the control eye after high-frequency irradiation (P<0.05). The expression of rhodopsin, ß-catenin, connexin36, calretinin, and calbindin in the retina of guinea pig declined. CONCLUSION: There is frequency cumulative damage effect on the retina that relates to LARP illumination frequency. This has significance for staff visual protection policies under LARP lighting conditions.

Therapeutic effect of a traditional Chinese medicine formulation on experimental choroidal neovascularization in mouse.

AIM: To investigate therapeutic effects of traditional Chinese medicine formulations, Hexuemingmu (HXMM) on laser-induced choroidal neovascularization (CNV) and follow-up effect in mice. METHODS: C57BL/6 mice of 8-week-old were used and CNV was induced with 577 nm laser photocoagulation. Animals were randomly divided into groups and different doses of HXMM were administered daily. One, four, and eight weeks after the intervention, the electroretinogram (ERG), fundus fluorescence angiography, choroidal flat mount and immunofluorescence staining were preformed to evaluate the function and CNV formation. The expression levels of angiogenic proteins were determined by Western blotting and immunofluorescence staining. An analysis of variance and Kruskal-Wallis test were used to test the differences among the groups. RESULTS: The results showed that HXMM effectively increased amplitude of ERG of mice (P<0.05), alleviated fundus CNV leakage (P<0.05), and reduced the area of neovascularization and the expression of angiogenic proteins (P<0.05) after laser-induced CNV. CONCLUSION: HXMM can protect the retinal function of mice after laser-induced CNV, and inhibit the CNV development.

Retinal neovascularization induced by mutant Vldlr gene inhibited in an inherited retinitis pigmentosa mouse model: an in-vivo study.

AIM: To explore whether the retinal neovascularization (NV) in a genetic mutant mice model could be ameliorated in an inherited retinitis pigmentosa (RP) mouse, which would help to elucidate the possible mechanism and prevention of retinal NV diseases in clinic. METHODS: The Vldlr -/- mice, the genetic mutant mouse model of retinal NV caused by the homozygous mutation of Vldlr gene, with the rd1 mice, the inherited RP mouse caused by homozygous mutation of Pde6b gene were bred. Intercrossing of the above two mice led to the birth of the F1 hybrids, further inbreeding of which gave birth to the F2 offspring. The ocular genotypes and phenotypes of the mice from all generations were examined, with the F2 offspring grouped according to the genotypes. RESULTS: The rd1 mice exhibited the RP phenotype of outer retinal degeneration and loss of retinal function. The Vldlr -/- mice exhibited the phenotype of retinal NV obviously shown by the fundus fluorescein angiography. The F1 hydrides, with the heterozygote genotype, exhibited no phenotypes of RP or retinal NV. The F2 offspring with homozygous genotypes were grouped into four subgroups. They were the F2-I mice with the wild-type Pde6b and Vldlr genes (Pde6b+/+ -Vldlr+/+ ), which had normal ocular phenotypes; the F2-II mice with homozygous mutant Vldlr gene (Pde6b+/+ -Vldlr-/- ), which exhibited the retinal NV phenotype; the F2-III mice with homozygous mutant Pde6b gene (Pde6b-/- -Vldlr+/+ ), which exhibited the RP phenotype. Specifically, the F2-IV mice with homozygous mutant Vldlr and Pde6b gene (Pde6b-/- -Vldlr-/- ) showed only the RP phenotype, without the signs of retinal NV. CONCLUSION: The retinal NV can be inhibited by the RP phenotype, which implies the role of a hyperoxic state in treating retinal NV diseases.

Application of Specific Fragment Length Polymorphism of Algae rDNA in Identification of Drowning Cases / 法医学杂志

OBJECTIVES@#To identify the drop-off location of victims in drowning cases, and confirm whether it is a fatal drowning or the victim is thrown into the water after death by detecting part of 5.8S sequence and second internal transcribed spacer （ITS2） （5.8S+ITS2） of diatom rDNA in water and organs.@*METHODS@#Two cases identified by diatom examination, which received by Nanjing Municipal Public Security Bureau Forensic Center, were taken as the research objects. The difference of the population structure of algae in water and human tissue was analysed by length polymorphism of 5.8S+ITS2 marker.@*RESULTS@#In case 1, similar species of diatom were detected from victim's lung and liver tissues and the water sample. Two kinds of DNA fragments with length of 330 bp and 376 bp were detected from victim's lung tissue and the water sample using 5.8S+ITS2 marker, which could confirm the victim was drowning before death. In case 2, there was no diatom found in victim's lung and liver tissues. Only one kind of DNA fragment with length of 331 bp and low relative fluorescence unit （RFU） was obtained from victim's lung tissue using 5.8S+ITS2 marker, thus the victim was thrown into the water after death.@*CONCLUSIONS@#The experimental results of the two cases in present study are consistent with the actual facts and the result of the diatom microscopic examination. The difference of population structure of specific microorganism in water and human tissue can be detected by 5.8S+ITS2 marker, which can help to identify the drop-off location of victims in drowning cases, and confirm whether it is a fatal drowning or the victim is thrown into the water after death.

Establishment and application of the demand-oriented service model for scientific research platforms in hospital / 中华医学科研管理杂志

Objective Promote the management and service of scientific research platforms and make sure their supporting role in hospital.Methods The demand of service object and service evaluation of scientific research platforms were obtained in Peking University People's Hospital,and the demand-oriented service mode for scientific research platforms was established and applied.Results With the implementation of this mode in hospital,the service strategies of scientific research platforms were optimized continuously,the awareness rate and service satisfaction were promoted in some extent.Conclusions This mode based on demand survey can promote the management and service of scientific research platforms in some extent and the better support and service can be provided for the sustainable development of scientific research in hospital.

Cytotoxic effects of γδT cells on bladder cancer cells and expression of MICA/B in bladder cancer / 北京大学学报(医学版)

OBJECTIVE@#To explore the role of γδT cells against bladder cancer and to detect the expression of stress proteins MICA/B recognized by γδT cells in bladder cancer.@*METHODS@#γδT cells from peripheral blood drawn from 6 bladder cancer patients with pamidronate stimulating were expanded. Flow cytometry was used to detect the purity and expansion folds of γδT cells, and the expression of CD107a on γδT cells after PMA/ionomycin stimulated. The cytotoxicity assay was carried out to test the cytotoxicity of γδT cells against human bladder cancer cell lines. The expression of MICA/B on bladder cancer cell lines and in bladder cancer tissues were detected through flow cytometry and immunohistochemistry respectively.@*RESULTS@#γδT cells from peripheral blood drawn from 6 bladder cancer patients were successfully expanded. The purity was 75%-94% and the expansion folds were 109-371 times. After being stimulated by PMA/ionomycin, the proportion of CD107a+ γδT cells increased significantly, reaching 40%-82%. γδT cells from the 6 bladder cancer patients showed obvious cytotoxic effects on 3 human bladder cancer cell lines which was enhanced as the effector: the target ratio increased. MICA/B were detected both in 3 bladder cancer cell lines and in 26 bladder cancer tissues. The staining score of MICA/B in invasive bladder cancer was slightly higher than that in non-invasive bladder cancer, and in advanced bladder cancer was higher than that in low grade bladder cancer, but the statistical analysis showed that the staining score of MICA/B was no significant correlation between the tissue and the tumor stages and grades.@*CONCLUSION@#γδT cells from the peripheral blood of the bladder cancer patients could be successfully expanded in vitro, and showed significant anti-bladder cancer effect. MICA/B were detected both in bladder cancer cell lines and in bladder cancer tissues. The statistical analysis showed that there was no significant correlation between the staining scores of MICA/B in the tissue and the tumor stages and grades.

Genetic Polymorphisms of 24 Y-STR Loci in Nanjing Han Population / 法医学杂志

OBJECTIVES@#To investigate the genetic polymorphism of DYS391 and other 23 Y-STR loci and to explore its application value in forensic science.@*METHODS@#Y-STRs loci of 580 unrelated Han males in Nanjing were amplified using AGCU Y-PLUS PCR （24） kit. The genetic parameters of 24 Y-STR loci such as gene frequency were calculated by software, and compared with the data of Hubei, Liao- ning, Guangdong, Beijing and Chengdu Han population.@*RESULTS@#Total 580 haplotypes were detected among 24 Y-STR loci in 580 unrelated Han males in Nanjing. The genetic diversity （GD） of each locus was from 0.294 6 to 0.939 8, and the haplotypes diversity （HD） was 0.983 7. There was a significant difference between the GD of 6 areas.@*CONCLUSIONS@#The 24 Y-STR loci such as DYS391 in Nanjing Han population have an application value in forensic science. They can also be used for cases testing and pedigree investigation.

G-protein coupled receptor 34 knockdown impairs the proliferation and migration of HGC-27 gastric cancer cells in vitro / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Overexpression of G-protein coupled receptor 34 (GPR34) affects the progression and prognosis of human gastric adenocarcinoma, however, the role of GPR34 in gastric cancer development and progression has not been well-determined. The current study aimed to investigate the effect of GPR34 knockdown on the proliferation, migration, and apoptosis of HGC-27 gastric cancer cells and the underlying mechanisms.</p><p><b>METHODS</b>The expression of GPR34 in gastric cancer cell line HGC-27 was detected by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. HGC-27 cells were employed to construct the stable GPR34 knockdown cell model in this study. Real-time RT-PCR and Western blotting were applied to validate the effect of short hairpin RNA (ShRNA) on the expression of GPR34 in HGC-27 gastric cells. The proliferation, migration of these cells were examined by Cell Counting Kit-8 and transwell. We also measured expression profile of PI3K/PDK1/AKT and ERK using Western blotting.</p><p><b>RESULTS</b>The ShRNA directed against GPR34 effectively inhibited both endogenous mRNA and protein expression levels of GPR34, and significantly down-regulated the expression of PIK3CB (P < 0.01), PIK3CD (P < 0.01), PDK1 (P < 0.01), phosphorylation of PDK1 (P < 0.01), Akt (P < 0.01), and ERK (P < 0.01). Furthermore, GPR34 knockdown resulted in an obvious reduction in HGC-27 cancer cell proliferation and migration activity (P < 0.01).</p><p><b>CONCLUSIONS</b>GPR34 knockdown impairs the proliferation and migration of HGC-27 gastric cancer cells in vitro and provides a potential implication for therapy of gastric cancer.</p>

Cytotoxic effects of mono-(2-ethylhexyl) phthalate on human embryonic stem cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Mono-(2-ethylhexyl) phthalate (MEHP), the metabolite of di-(2-ethylhexyl) phthalate (DEHP), was suspected to be toxic to human embryos. This study contributes to investigating its toxic effects by an embryonic stem cell test (EST) based on two human embryonic stem cell (hESCs) lines.</p><p><b>METHODS</b>CH1 established in our own lab and H1, a federally registered cell line were two human embryonic stem cell lines used in this test. Four endpoint measurements were performed consisting of cell viability, proliferation ability, apoptosis as well as changes of gene expression patterns after spontaneous differentiation were determined. For measuring effects on the first three endpoints, the cells were treated with various concentrations of MEHP dissolved in dimethyl sulfoxide (DMSO) and only with DMSO which served as control and harvested after 5 days. For measuring effects during spontaneous differentiation, the RNA of embryoid bodies (EBs) formed after 8 days' MEHP exposure was collected and changes in differentiation specific gene expression patterns were analyzed by quantitative real time RT-PCR.</p><p><b>RESULTS</b>As a result the viability and proliferation ability of both cell lines decreased significantly at 1000 µmol/L MEHP, while there was no effect on apoptosis or cell morphology. In addition MEHP also changed the gene expression pattern in the EBs of both cell lines.</p><p><b>CONCLUSION</b>MEHP in a high dose was cytotoxic and affected the development of hESCs, which indicates its embryo toxicity in human embryos.</p>

Overexpression or knock-down of runt-related transcription factor 1 affects BCR-ABL-induced proliferation and migration in vitro and leukemogenesis in vivo in mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Runt-related transcription factor 1 (Runx1) plays a crucial role in hematogenesis and its dysfunction may contribute to leukemogenesis. However, it is not clear whether or not abnormal expression of Runx1 will induce leukemia and how the change of Runx1 expression level could affect BCR-ABL-induced leukemogenesis. In the present study, we aimed to analyze if abnormal expression of Runx1 in BaF3 cells alone would induce leukemogenesis. And we also wanted to know if abnormal expression of Runx1 in leukemic cells would affect leukemogenesis. Furthermore, we investigated whether overexpression or knock-down of Runx1 in BaF3 cells would induce leukemogenesis.</p><p><b>METHODS</b>Plasmids containing full-length Runx1 cDNA were transduced into BaF3 cells and BaF3-P185wt cells (BCR-ABL transformed BaF3 cells) by electroporation. Plasmids containing a short hairpin RNA of Runx1 were transduced into BaF3 cells and BaF3-P185wt cells by electroporation. Runx1 expression level was quantified by Western blotting and quantitative real-time PCR. The effects of overexpression or knock-down of Runx1 on proliferation, apoptosis and migration of cells were detected in vitro. Then, using MSCV-P185wt-EGFP as a control, we transplanted MSCV-P185wt-Runx1 cells or MSCV-P185wt-shRNA cells into Balb/c mice through tail vein and observed tumorgenesis of the different phenotypes.</p><p><b>RESULTS</b>In vitro analysis revealed that overexpression of Runx1 in P185wt cells could inhibit cell proliferation and slow down cell migration; while knock-down of Runx1 could promote cell proliferation and speed up cell migration. In vivo analysis indicated that mice transplanted with MSCV-P185wt-Runx1 survived longer than controls. In contrast, mice transplanted with MSCV-P185wt-shRNA survived shorter than the control group. Gross pathological analysis revealed that the MSCV-P185wt-Runx1 group had less severe splenomegaly and hepatomegaly compared to the control group, and the MSCV-P185wt-shRNA group had more severe splenomegaly and hepatomegaly. No splenomegaly or hepatomegaly was detected in mice transplanted with MSCV-BaF3-Runx1 cells or MSCV-BaF3-shRNA cells. Both the mice of MSCV-BaF3-Runx1 group and MSCV-BaF3-shRNA group were healthy with no sign of leukemia for up to three months.</p><p><b>CONCLUSIONS</b>Overexpression or knock-down of Runx1 gene in BaF3 cells alone could not induce leukemogenesis. However, in BaF3-P185wt cells, alteration of Runx1 expression could affect BCR-ABL-induced proliferation and migration in vitro and leukemogenesis in vivo.</p>

STR genotyping from trace epithelial cells on fountain pen / 法医学杂志

OBJECTIVE@#To evaluate the feasibility of STR genotyping from trace epithelial cells on fountain pen and to discuss the impact of conservation time on DNA typing.@*METHODS@#Seven fountain pens were separately used by each of the 17 volunteers 20 minutes per day for a month and then were preserved on day 1, 3, 5, 7, 14, 21, and 28. DNA was extracted from the epithelial cells on fountain pen by silicon bead and was genotyped by Identifier kit. The corresponding control samples were buccal swabs of the above volunteers. The detectable numbers of loci were counted for assessment.@*RESULTS@#There were statistically significant differences in the DNA genotyping by detectable numbers of gene loci between buccal swabs and epithelial cells on fountain pen of different conservation times (P < 0.01). The differences of detectable numbers of loci between the epithelial cells on fountain pen preserved on day 1, 3, 5, 7, 14, 21, 28 and the corresponding oral swabs were also statistically significant (P < 0.01). More than 12 loci could be successfully genotyped in 41.2% samples from the epithelial cells on fountain pen if the tests were performed within 24 hours.@*CONCLUSION@#The trace epithelial cells on fountain pen can be used as biological samples for personal identification, but the conservation time would have influence on the results of DNA genotyping.

Localization of cystathionine beta synthase in mice ovaries and its expression profile during follicular development / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>In vitro fertilization (IVF) researches have suggested that cystathionine beta synthase (CBS) is involved in oocyte development. However, little is known about the regional and cellular expression patterns of CBS in the ovary. The purpose of this study was to analyze the localization of CBS in mice ovaries and to investigate the expression profile during follicular development.</p><p><b>METHODS</b>We used in situ hybridization and immunohistochemical analysis to determine CBS expression in the ovaries of female Balb/c mice. Then the follicles were collected from F1 (C57BL x Balb/c) mice and cultured in vitro. With the method of semi-quantitative RT-PCR, we also investigated the expression profile of CBS during follicular development.</p><p><b>RESULTS</b>CBS was absent in the oocytes, although it was ubiquitously expressed in the ovary with the strongest expression in follicular cells at all stages. In late antral follicles, CBS expression was markedly higher in granulosa cells located close to the antrum and in cumulus cells around the oocyte. The semi-quantitative RT-PCR showed that CBS mRNA was detected in follicles at all stages in vitro. In cumulus-oocyte complexes superovulated, CBS expression also increased rapidly.</p><p><b>CONCLUSIONS</b>CBS was located mainly in the follicular cells in the ovaries. The level of CBS expression is high in follicles during folliculogenesis in mice. Differences in the CBS expression profile between oocyte and follicular cells suggest a role for CBS as a mediator in interactions between oocyte and granulosa cells.</p>

Epigenetic modifications and its impact on animal cloning / 生物工程学报

Despite recent successes in cloning various mammals and amphibians, the low efficiency of animals production and abnormal symptoms in many cloned animals are crucial problems in cloning technology. To overcome these problems, scientists focus on mechanisms of cloning. A possible cause of the low success frequency of cloning is the insufficient dedifferentiation and the inadequate reprogramming of the high differentiated adult somatic nucleus in enucleated oocytes, which caused by incomplete methylation and premature de novo remethylation of donor DNA. In cloned embryos the methylation level is higher than normal embryos, and this may cause aberrant expression of several important genes, especially imprinting genes. Study on these mechanisms is very important to improve the rate of successful cloned animals.

Isolation and identification of reprogramming genes related to the development of the rabbit reconstructed embryos / 生物工程学报

By the method of single preimplantation embryos differential display polymerase chain reaction (SPEDDRT-PCR), 25 reprogramming cDNA fragments were obtained from single 2-cell, 8-cell embryos and blastula. After cloning and sequencing, five of them were identified by reverse-Northern and characterized with stage-specific expression during reconstructed embryo development. This results will help to isolate full length reprogramming genes and study their function during embryonic development.