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BACKGROUND: Researchers believe that hydrogen sulfide (H2S), as an important cell protective molecule, may become a new treatment method to restore the physiological function of diseased cells or organ systems through the artificial regulation of endogenous H2S biosynthesis or in vitro administration of H2S donor. ADT-OH is a slow-release donor of H2S that can improve the survival rate of hippocampal nerve cells with glutamate-induced injury, but studies on the proliferation of cerebral cortical neural precursor cells are rare. OBJECTIVE: To investigate the effect of ADT-OH on the proliferation of neural precursor cells in embryonic cerebral cortex. METHODS: Neural precursor cells from cerebral cortical ventricular zone and subventricular zone of embryonic mice at embryonic 14.5 days were isolated. Neural precursor cells from one fetal mouse were inoculated into one well (24-well plate), and cultured with the medium containing 100 μmol/L ADT-OH. The size and number of neural spheres per well were measured at 3 days after culture. The proliferation rate of cultured neural precursor cells was detected by BrdU labeling. The proliferation of the cells was further verified by immunofluorescence staining with the specific antibody Ki67. The expression of cyclin D1 was finally detected by western blot assay. RESULTS AND CONCLUSION: Our experimental results showed that ADT-OH could promote the formation of neural spheres, and further detection by BrdU and Ki67 antibody showed that ADT-OH could promote the proliferation rate of neural precursor cells. Meanwhile, the expression of cyclin D1, a proliferation-related gene, was up-regulated in neural precursor cells after ADT-OH treatment. Overall, ADT-OH may promote the proliferation of neural precursor cells by regulating the expression of cyclin D1.
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BACKGROUND: Studies have shown that Lycium barbarum polysaccharide (LBP) has the functions of anti-aging, nerve protection, anti-fatigue, blood sugar control, anti-oxidation, and anti-tumor. It may have some protective effects against osteoarthritis of the knee, but have been rarely reported. CD151 and matrix metalloproteinase 3 (MMP-3) are two common cytokines for assessing knee osteoarthritis. OBJECTIVE: To observe the effect of LBP on the expression of CD151 and MMP-3 in rabbit osteoarthritis. METHODS: Sixty-four healthy 6-month-old white rabbits were randomly divided into four groups: blank group, model group, LBP group and normal saline group. Animal models of knee osteoarthritis were made using Hulth method in the rabbits except those in the blank group. The rats in the LBP and normal saline groups were fed with normal dose of LBP and normal saline for 4 weeks, and then the articular cartilage tissues were taken from the affected side at 12 weeks after modeling. The morphological changes of the articular cartilage were observed by hematoxylin-eosin staining. The expression levels and spatial distribution of CD151 and MMP-3 in articular cartilage was observed by immunohistochemical staining and western blot. Ethic approval was given by the People’s Hospital of Ningxia Hui Autonomous Region (approval No. 2014-30817). RESULTS AND CONCLUSION: immunohistochemistry staining and western blot results showed that the absorbance values and protein expression of MMP-3 and CD-151 were significantly lower in the LBP group than the normal saline and model groups (P < 0.05). Therefore, the expression of CD151 and MMP-3 in the articular cartilage of osteoarthritis was increased, and LBP could inhibit the expression of CD151 and MMP-3 in osteoarthritis, so as to slow down the occurrence of osteoarthritis.
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Objective@#To elaborate the characteristics and advantages of Whole-Mount immune fluorescence staining by observing the lymphatic vessels of mice.@*Methods@#The ear skin tissue, the hindlimb lymphatic vessels and the mesenteric lymphatic vessels were harvested from normal C57 mice. The tissue samples were subjected to whole-tissue immunofluorescence staining.These tissue samples were fixed by paraformaldehyde, blocked by bovine serum and incubated in primary and secondary antibodies. Then, the lymphatic vessels were observed and analyzed in these samples with a confocal laser-scanning microscope.@*Results@#The capillary lymphatic vessels and lymphatic endothelial cells can be clearly showed in the ear skin. The valves and smooth muscles can be clearly showed in the hindlimb and mesenteric lymphatic vessels by Whole-Mount immunofluorescence staining.@*Conclusions@#The whole-tissue immunofluorescence staining technique can observe the external morphology of lymphatic vessels clearly and stereoscopically, and can deeply observe the internal structure of lymphatic vessels. This technique can provide more accurate study on physiology and pathology of lymphatic vessels.
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Objective To make quantitative analysis on collected cell images combined with machine learning integrated clustering algorithm, so as to explore a method for fast recognition and classification of cells in mixed cultures based on morphology. Methods The morphometric properties of A549 and 3T3 cells in vitro were characterized by immunostaining, the fluorescent images were then analyzed with CellProfiler to extract the parameters of cell morphology. The parameters were loaded into CellProfiler Analyst to be trained with machine learning algorithm, and a rule was developed to form a generalization capability for cell classification in mixed cultures. Results The accuracy of the training classifier was 81-24%, and the binary classifications of A549 and 3T3 cells could be realized. Conclusions The method of machine learning is very effective in parameter clustering. The application of machine learning into cell image recognition can provide pre-judgment for rapid pathological examination of tissue sections, thereby reducing the workload of doctors and improving the accuracy of diagnosis.
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Objective@#To compare the indirect immunofluorescence staining effect of urinary podocytes by thinprep liquid-based cytologic test (TCT) with that by the conventional centrifugal smear method, and explore its clinical application value. @*Methods@#The morning urine samples from 50 patients with type 2 diabetic nephropathy and 14 healthy controls were smeared with the TCT and conventional centrifugation method, respectively, and then the indirect immunofluorescence staining were performed to observe the morphology of podocytes. @*Results@#For 64 urine samples, the satisfactory rate of TCT smears (85.94%) was significantly higher than that of conventional smears (50.00%), and the podocyte detection rate of TCT smears (73.47%) was also significantly higher than that of conventional smears (51.02%) (P＜0.05). When urinary podocytes of the same patient were positive by both methods, the reading effect of TCT smears was obviously superior to that of conventional smears. @*Conclusion@#The TCT combined with indirect immunofluorescence staining is obviously superior to the conventional centrifugal smear method in the podocyte diagnosis of urine samples.
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Objective Clinical, pathological and molecular biological data of six cases with molecular diagnosis of collagen type Ⅵ related myopathies were retrospectively analyzed to improve the recognition of collagen protein Ⅵrelated myopathy.Methods Clinical and pathological data of six patients diagnosed as collagen protein Ⅵ related myopathy by next generation sequencing and molecular biologic analysis during 2010-2017 were summarized.Results All of the six patients presented early childhood onset ((2.00 ±0.75) years old), and delayed motor growth after birth.There were six cases of proximal muscle weakness , one with distal muscle weakness; three cases with osteoarthropathy; one case of severe skin scar.The creatine kinase levels (187-380 U/L) of three patients were slightly elevated .Three cases showed myogenic damage , two with mild neurogenic lesions , one with myogenic and neurogenic damages . Next generation sequencing showed four cases with COL 6A1 gene heterozygous mutation ( a novel mutation and three had been reported), one with COL6A2 heterozygous mutation and one with COL6A1 and COL6A2 complex heterozygous mutation .The pathological analysis of skeletal muscle biopsy showed that the muscle fiber size was different , and the connective tissue elements were seriously increased .Some opaque fibers were observed.Six cases were found anti-collagen Ⅵ/Ⅳ protein monoclonal antibody immunofluorescence double stained , and sarcolemma Ⅵcollagen protein expression decreased to different degrees .Conclusions The clinical manifestations of the collagen protein Ⅵ related myopathy were found to be complex .Skeletal muscle biopsy pathological analysis was lack of specificity . Anti-collagen Ⅵ/Ⅳ monoclonal antibody immunofluorescence double staining showed collagen protein Ⅵ missing partly or completely . Immunofluorescence staining and the next generation sequencing can improve the diagnosis of collagen proteinⅥrelated myopathy.
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Objective:To observe the characteristics of the tissue distribution of the blood brain barrier through the modification of the liposome encapsulated brain derived neurotrophic factor.Methods: A total of 32 SD rats were selected from Xi′an Medicine College of Foreign Affairs as the research object,and randomly divided into two groups (n=16) and control group (n=16).To establish a rat model of AD rats in the control group received no treatment,liposome group by rat tail vein injection of 10 μg/kg Tf-pGFAP-BDNF-PEG,analysis of liposome entrapped BDNF targeting liposomes through blood brain barrier tissue distribution characteristics.Results: 40-50 nerve growth factor (1 ml) was collected by using 24mCi99mTC and 20 L 0.5 ml was collected on the top of the column.The concentration of 8-17 tube in the peak was recovered,and the results showed that the labeling rate of neurotrophic factor was 98.9％.Liposome group for tenth days,14 days of BDNF immunoreactive cells,significantly higher than the control group (P<0.05);neurotrophic factor in brain tissue of rats in body lipid gene expression at day fourteenth,significantly higher than the control group (P<0.05);expression of neurotrophic factors in liposome group rat,and neurotrophic factor expression level was significantly higher than the control group (P<0.05).The cerebrospinal fluid was clear,colorless and transparent,and no red blood cells were detected under microscope.Liposome group in cerebrospinal fluid of rats with fecal occult blood detection results of gold colloid showed negative,while the control group was positive.Conclusion: The combination of transferrin and polyethylene glycol modified liposomes combined with brain specific promoter can improve the targeting of liposomes.
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Objective To explore the cellular localization of chemokine (C-X-C motif) ligand 1 (CXCL1) in brain tissue and its expres-sion in brain tissue and blood in patients with severe traumatic brain injury (TBI), as well as its correlation with the injury severity. Methods From September, 2013 to October, 2015, 78 cases of TBI with craniotomy admitted to our hospital were involved as TBI group. A total of 78 peripheral blood samples and 19 brain tissue samples were studied. According to the scores of Glasgow Coma Scale (GCS) at admission, the TBI group was classified as severe TBI group (6~8, n=35) and particularly severe TBI group (3~5, n=43). Ten cases of control brain tissue were taken from patients with cerebral aneurysms or benign tumor and also undergoing craniotomy during the same time. Peripheral blood from ten healthy people were involved as the healthy control group. Immunofluorescent double staining was used to detect the cellular local-ization of CXCL1 in brain tissues, and ELISA was used to detect the expression of CXCL1 in brain tissue and blood. The relationship be-tween the level of CXCL1 in peripheral blood at different time and the score of Glasgow Outcome Scale (GOS) was analyzed with Spear-man correlation analysis. Results In normal brain tissue, CXCL1 mainly localized in astrocytes. For severe TBI, CXCL1 mainly expressed in neurons and astrocytes. The level of CXCL1 was higher in brain tissue in the particularly severe TBI group than in the severe TBI group (t=-12.58, P0.05). In the particularly severe TBI group, the level of CXCL1 in blood reached a peak before and one day after surgery, then gradually decreased, and was still higher than that in the healthy control group 30 days after surgery (P<0.05). The level of CXCL1 in blood was higher in the particularly severe TBI group than in the severe TBI group at all time points (P<0.05), and the level before surgery was negatively correlated with the score of GOS in the particularly severe TBI group (r=-0.351, P<0.05). Conclusion The CXCL1 protein of injury brain tissue was mainly colocalized in neurons and astrocytes in severe TBI patients, and the ex-pression was associated with injury severity and outcome.
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Objective To investigate the effects of prostaglandin E2(PGE2) in prostate mesenchymal cell activation .Methods To culture human prostate stromal cells WPMY‐1 in vitro ,implement immunofluorescence staining after treating with DMSO and 10-9 mol/L PGE2 and detect the change of the myofibroblast phenotype .The expression of cellular inflammatory factor interleukin 8(IL‐8) was detected by real‐time quantitative PCR and the concentration of IL‐8 was detected .Results PGE2 significantly in‐creased the cellular proportion of colocalization with alpha SMA and Vimentin in WPMY‐1 cells .PGE2 promoted the expression of IL‐8 in WPMY‐1 cells .Conclusion PGE2 can increase myofibroblast phenotype in human prostate mesenchymal cells WPMY‐1 in vitro culture ,promotes the IL‐8 expression ,which has an important role in the occurrence and development of prostate cancer .
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Objective To assess the expression of the corneal epithelial TJ protein claudin -1 in type 2 diabetic rats at different time points.Methods 80 eight-week-old male SD rats were randomly divided into the normal control and diabetic mellitus(DM) groups(n =40 each).A high-fat diet combined with STZ injection was used to induce type 2 DM.Normal and diabetic rats were sacrificed at 4, 8, 16 weeks respectively(after STZ injection) before de-bridement.Hematoxylin and eosin was used to study the morphological differences between normal and diabetic cor -nea.Immunofluorescene and Western blot were used to determine the expression of corneal epithelial TJ protein claudin-1.Results Corneal epithelial cells reduction and stroma edema were evident at 8 and 16 weeks by HE. Claudin-1 expression in the corneal epithelium of the diabetic group was lower and fainter compared to the normal group at 16 weeks(after STZ injection)(P <0.05), which were similar to the normal group at 4 and 8 weeks.Con-clusion Continual hyperglycemia has a negative effect on ocular surface tissues and the expression of corneal epi -thelial tight junction protein claudin -1 with progressing of type 2 diabetic mellitus.
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Objective To establish an experimental model for the study of α-particle-induced bystander effect of DNA damage and investigate the characteristics of bystander DNA double-strand break (DSB).Methods The red fluorescence fusion protein of HsBrkl-RFP was used to mark the cytoplasm of one cell line to distinguish the irradiated target cells (HFS-RFP) and the non-irradiated bystander cells (HFS) in the co-culture cellular model.After α-particle irradiation,cellular DSB and its repair kinetics were analyzed by the immunofluorescence staining of γH2AX and laser confocal microscope observation.Results A bystander studying model was established by co-culturing human HFS-RFP cells with its partner HSF cells.After 0.1 Gy or 0.2 Gy α-particle irradiation,the similar kinetics of γH2AX foci production and abatement were observed in both irradiated HFS-RFP cells and non-irradiated bystander HFS cells,in which the highest level of γH2AX foci was detected at 1 h post-irradiation.The second peak of γH2AX foci formation appeared at 8 h post-irradiation,which possibly indicates the occurrence of secondary DSB.However,the production of secondary DSB in the bystander cells was weaker than that in the irradiated cells.Conclusions The cell co-culture model can be used for bystander effect investigation.Bystander DSB can be effectively induce by irradiation and the secondary breakage of DNA DSB in the bystander cells may relative to the consequential biochemical processing of clustered DNA damage.
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Objective To establish a human umbilical artery EC-SMC co-culture model, and mimic the morphological and functional characteristics of human arterial wall, for further reseach of the pathological mechanism and therapy of atherosclerosis and imflammatory damage. Methods We secceeded in the primary culture of human umbilical artery endothelial cells (HUAEC) and human umbilical artery smooth muscle cells (HUASMC) by collagenase perfusion digestion and tissue planting, respectively. HUASMCs were incubated in a medium with ascorbic acid at the concentration greater than 50 μg/mL to produce collagen, which was considered as the extracellular matrix for ECs. Then HUAECs were seeded directly upon HUASMCs in a saturate density for sufficient direct physical interaction between ECs and SMCs. The morphological characteristic of EC-SMC co-culture was identified by immunofluorescence staining, and the function of EC-SMC co-culture was identified by Dil-Ac-LDL uptake test. Results The morphological identification showed that the entire surface of HUASMCs was covered by a confluent monolayer confluent monolayer, which indicated that the model had simulated the morphological characteristic of human arterial wall. The results of Dil-Ac-LDL uptake test showed that there was a fluorescent signal in HUAECs. Compared with EC monoculture, the Dil-Ac-LDL uptake of HUAECs was increased significantly in the co-culture system. All the reseach results indicated that there was an interaction between HUAECs and HUASMCs in the co-culture system. Conclusions In the present study, human umbilical artery EC-SMC co-culture model was constructed successfully, which could mimic the morphological characteristic and basic functions of human arterial wall.
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Objective Applying a reliable precise method to assess the mitotic index of cardiomyocytes,to disclose of disclosure the mechanism implicated in cardiomyocytes proliferation. Methods H9c2(2-1) cardiomyocytes were originally developed from rat BD1X heart(ATCC).These cells were cultured on coverslips.Double immunofluorescence staining with monoclonal antibodies(1:100) against phospho-histone H3 and ?-sarcomeric actin was performed on the cultured cells.Anti-mouse IgG FITC was used as the secondary antibody for the H3P antibody,and anti-mouse IgM Cy3 was used as the secondary antibody for the ?-sarcomeric actin.DNA was visualized with Hochest 33342.All photographs were taken with an Olympus fluorescence microscope. Results The cytoplasm of cardiomyocytes appeared red,the mitotic chromosomes green with distinct shape,and Hochest 33342-stained nuclei blue.Conclusion Our method is the reliable and exact means to observe and assess cardiomyocytes mitosis.
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Glia maturation factor(GMF)detected in the adult brain is an acidic proteinthat has the ability to promote the morphological and chemical differentiation ofastroblasts,but this effect is reversible.In the present study we have establishedneuron-enriched dissociated primary cultures from 7-day-old rat cerebellar cortexin which about 97% of the cells are neurons especially granule cells using differential count of indirect immunofluorescence.The addition of purified GMF to the culturesmarkedly enhances neuronal survival,while control cultures grown in the absence ofGMF exhibit a significant decrease in neuronal number.These GMF effects aredose-dependent,with optimal stimulation occurring at a concentration of 250 ng/ml.Although the mechanism by which purified GMF influences cerebellar corticalneuronal survival is not known,these results suggest that GMF not only affectsglial cells,but also may function as a neurotrophic agent in the central nervousmetsys