Objective To explore the effects of Neurogenesin 1 (Ng1) gene on functional recovery after spinal cord injury (SCI) and its mechanism. Methods Thirty-six rats (aging 4 months, weighing 230 g and being male or female), were randomly divided into two groups: experimental group (n=18) and control group (n=18). After spinal cord contusive injury at T10 level was made in all these rats using modified Allen’s method, Ng1 recombinant plasmid and blank plasmid were transfectedinto the damaged areas of exprimental group and control group respectively by Alzet pumps. At 1 day, 1 week, 2 weeks, 3 weeks, and 4 weeks after SCI, Basso-Beattle-Bresnahan (BBB) Rating Scale was used to observe the recovery of motor function. At 1 week after injury, the expressions of Ng1 mRNA and protein in injured spinal cord were detected by RT-PCR and Western blot techniques. And at 2 and 4 weeks, double immunofluorescence and histopathologic examinations were performed to study the prol iferation of the adult endogenous neural stem cells and pathological change after SCI. Results At 1-4 weeks after SCI, the BBB scores in the exprimental group was significantly higher than that in control group (P lt; 0.05), and at 4 weeks the BBB score of the experimental group (16.80 ± 1.79) was significantly higher than that of the control group (9.60 ± 1.67), (P lt; 0.01). RTPCR and Western blot showed that the mRNA and protein expressions of Ng1 were observed in the exprimental group and no expression was seen in the control group. Histologic observation showed that the morphology of spinal cord and neurons in the exprimental group was better than that in the control group and was close to the normal tissue. The mean number of Nestin+/ BrdU+ newborn endogenous neural stem cells in the exprimental group was significantly more than that in control group (P lt; 0.05). Conclusion Ng1 gene could promote the prol iferation of endogenous neural stem cells and protect the injured neurons, which enhances the repair of the motor function after SCI.
Epigenetic modifications such as DNA methylation, histone post-translational modifications, non-coding RNA are reversible, heritable alterations which are induced by environmental stimuli. Major risk factors of diabetes and diabetic complications including hyperglycemia, oxidative stress and advanced glycation end products, can lead to abnormal epigenetic modifications in retinal vascular endothelial cells and retinal pigment epithelium cells. Epigenetic mechanisms are involved in the pathogenesis of macular edema and neovascularization of diabetic retinopathy (DR), as well as diabetic metabolic memory. The heritable nature of epigenetic marks also playsakey role in familial diabetes mellitus. Further elucidation of epigenetic mechanisms in DR can open the way for the discovery of novel therapeutic targets to prevent DR progression.
ObjectiveTo identify and observe disease-causing gene variants and clinical phenotypes in a Han Chinese family with Leber congenital amaurosis (LCA). MethodsA retrospective study. A patient with LCA10 and his parents who had presented at Department of Ophthalmology of Henan Provincial People's Hospital on May 2022 were selected as the study subject. Detailed medical and family histories were recorded, fundus photography and flash electroretinogram (F-ERG) were performed. Peripheral venous blood samples (3 ml) of the proband and his parents were collected to extract whole genomic DNA, then whole exome sequencing (WES) and mitochondrial DNA (mtDNA) sequencing were carried out for the proband to determine the disease-causing gene and variants. All variants were annotated by bioinformatics analysis. According to the American College of Medical Genetics and Genomics (ACMG) guidelines, the pathogenicity of all detected variants were evaluated. Candidate variants were verified by Sanger sequencing, and in vitro minigene assay were performed to evaluate the impact of the missense variant with insufficient evidence on mRNA splicing. ResultsThe proband, male, 7-month-old, presented with an inability to follow light or objects, eye poking, photophobia, nystagmus, partial loss of retinal pigment epithelium around the fovea of the macula. At the age of 2 years old, F-ERG revealed severe reduction, elongation, or even no waveform of a-wave and b-wave in both eyes. No obvious abnormality was found in the clinical phenotype of his parents. The result of WES revealed that the proband carried two variants in exon 40 and exon 2 of CEP290, a frameshift variant c.5515_5518del (p.Glu1839Lysfs*11) (V1) and a novel missense variant c.74C>T (p.Ala25Val) (V2), respectively. The result of mitochondrial DNA sequencing was negative. Sanger sequencing confirmed that the heterozygous frameshift variant was inherited from his father and the heterozygous novel missense variant was inherited from his mother, which constituted compound heterozygous variants. In vitro minigene splicing assay confirmed that V2 created a new splicing donor at exon 2, leading to the in-frame deletion of 30bp fragment during transcription and loss of 10 amino acid residues in the protein. The two variants were pathogenic (V1) and likely pathogenic (V2) based on ACMG guidelines, respectively. ConclusionsThe c.5515_5518del and novel c.74C>T compound heterozygous variants of the CEP290 gene probably are the cause of LCA10 in this family, which lead to the production of a truncated protein and aberrant splicing of pre-mRNA, respectively. LCA is characterized by early onset, severe impairment of visual function, and a wide range of disease-causing variations.
Objective lt;brgt;To evaluated the effect of transpupillary thermotherapy (TTT) on age-related macular degeneration (AMD). lt;brgt; lt;brgt;Methods lt;brgt;Sixty-two cases (62 eyes) of exudative AMD were managed with TTT. Before treatment, 58 cases underwent fundus fluorescein angiography(FFA),42 cases underwent simultaneous indocyanine green angiography (ICGA), and 56 cases underwent optic coherence tomography (OCT).TTT was delivered using a 810 nm diode laser with variable spot sizes 0.5-3.0 mm and power range 60-40 mW,60 seconds duration. Sixty-two cases were followed up for 1-10 months with 4.8 months average. lt;brgt; lt;brgt;Results lt;brgt;The visual acuities of last visit were compared with those before the treatment. The visual acuity was unchanged in 43 cases (69.3%), improved in 15 cases (24.2%), and declined in 4 cases (6.5%). OCT was re-done in 51 cases and compared with OCT images before TTT treatment. The height of macular edema was unchanged in 29 cases (56.9%), decreased in 18 cases (35.3%), and increased in 4 cases (7.8%). The amelioration of visual acuity was compatible with that of macular configuration in the majority of cases (74.5%). Only in 13 cases (25.5%) the amelioration of visual acuity lagged behind that of macular configuration. The re-treatment was performed in 18 cases (29.1%), probably due to insufficiency of laser power. No side-effect was found. lt;brgt; lt;brgt;Conclusion lt;brgt;TTT makes most of the cases of exudative AMD retaining or improving their visual acuity. The employment is secured. Further exploration is needed in order to obtain the parameters of the laser treatment. (Chin J Ocul Fundus Dis, 2002, 18: 180-183)
Objective To summarize and review the heterogeneity of bone marrow derived stem cells (BMDSCs) and its formation mechanism and significance, and to analyze the possible roles and mechanisms in intestinal epithel ial reconstruction. Methods The related l iterature about BMDSCs heterogeneity and its role in intestinal epithel ial repair was reviewed and analyzed. Results The heterogeneity of BMDSCs provided better explanations for its multi-potency. The probable mechanisms of BMDSCs to repair intestinal epithel ium included direct implantation into intestinal epithel ium, fusion between BMDSCs and intestinal stem cells, and promotion of injury microcirculation reconstruction. Conclusion BMDSCs have a bright future in gastrointestinal injury caused by inflammatory bowl disease and regeneration.
In order to explore further the regulatory factors to the potentiality in inducing osteogenesis by fibroblasts, the fibroblasts were isolated, and purified from human skin, and were grown in incubation in the media of EGF, IL-6, TNF-alpha and BMP2 at different concentrations for two weeks, then, the markers for osteogenic features were investigated by biochemistry, histochemistry and electron microscopic observations. It was found that the combined use of TNF-alpha and BMP2 could stimulate fibroblasts to secrete alkaline phosphatase, osteocalcin and collagen, and the morphological changes of the fibroblasts were also very striking. In the extracellular matrix, the collagen fibrils, with or without periodicity, were arranged regularly or randomly oriented, and numerous minute calcium granules were interspersed among them. The fibroblasts were interwoven one on top of another in the form of multilayer structure and on the surface, there were secreting granules and piling up of calcium crystals which coalessed steadily and increased in size in forming bony nodules. It was considered that TNF-alpha and BMP2 were capable of inducing the fibroblasts to form bone.
OBJECTIVE To investigate the ectopic osteogenesis of bone marrow stromal cells (MSC) induced by bone morphogenetic protein(BMP) in vitro and in vivo, providing the experimental evidence for making an artificial bone with its own capacity of bone formation. METHODS MSC were separated and cultured from bone marrow of Wistar rats, MSC were co-cultured with BMP in vitro (cultured in plate and diffuse chamber). Artificial coral hydroxyapatites (CHA) with MSC and BMP were implanted into dorsal muscles of Wistar rats, their bone formation were observed by morphological examination, histochemistry and immunohistochemistry. RESULTS Only cartilaginous matrix were produced by MSC in vitro (cultured in plate and diffuse chamber), and both cartilaginous and bone matrix production within the combined grafts were seen. The bone formation of experimental groups (CHA + BMP + MSC) was ber than that of control A(CHA + MSC) and control B(CHA). CONCLUSION It may be possible to produce an artificial bone with its own capacity of bone formation by combined graft (CHA + BMP + MSC). There may be multiple factors as well as BMP inducing bone formation both in the whole body and the location of the implantation. Further research on these factors will have the significance for making the ideal artificial bone.
摘要:目的:研究高血压病患者过氧化物酶体增殖物激活受体(PPAR)γ2基因Pro12Ala多态性与血糖水平之间的关系。方法:纳入177名原发性高血压患者,其中空腹血糖(FBG)lt;5.6 mmol/L组65例, FBG≥5.6 mmol/L组112例,收集一般资料;分别测定空腹及餐后2小时血糖、胰岛素;对PPARγ2 基因Pro12Ala多态性与各临床变量的关系进行研究。结果:FBGlt;5.6 mmol/L组和FBG≥5.6 mmol/L组Pro和Ala等位基因频率分别为0.333,0.034及0.602,0.031;PP和PA基因型频率分别为0.299,0.068及0.571,0.062;无AA型纯合子。以体重指数(BMI)分层后,BMIlt;25组内,FBG与PPARγ2基因型相关（P=0.029)。以基因型分组比较,PA组空腹血糖水平和胰岛素抵抗指数都低于PP组(Plt;0.05)。结论:成都地区高血压患者PPARγ2基因Pro12Ala多态性与空腹血糖水平相关,且携带Ala基因者空腹血糖水平较低,胰岛素抵抗较轻,推测该突变可能有减轻高血压病患者胰岛素抵抗,改善糖代谢异常的作用。Abstract: Objective:To study the association between the Pro12Ala polymorphism in peroxisome proliferatorsactivated receptorγ2 ( PPARγ2 ) gene and blood glucose levels in patients with primary hypertension. Methods：The Pro12Ala polymorphism in PPARγ2 was determined by polymerase chain reactionrestriction fragment length polymorphism (PCRRELP) in 177 subjects with primary hypertension of the Han people in Chengdu of China, including 65 subjects with fasting blood glucose (FBG)lt;5.6 mmol/L and 112 subjects with FBG≥5.6 mmol/L; the clinical characteristics including height, weight, OGTT(0h and 2h) of the subjects were detected and the realationship between the Pro12Ala polymorphism and the clinical characteristics were analysed. Results: The allele frequencies in the group with FBGlt;5.6 mmol/L and FBG≥5.6 mmol/L were 0.333, 0.602 for Pro and 0.034, 0.031 for Ala. The genotype frequencies were 0.299, 0.571 for PP and 0.068, 0.062 for PA, and there was no AA. In the group with BMIlt;25, the Pro12Ala polymorphism was associated with FBG （P=0.029). the Ala allele had a negative relationship to the FPG and insulin resistance index (IRI) (Plt;0.05).Conclusion: The data showed that the Pro12Ala polymorphism was associated with FBG., and The allele Ala probably had benefits to glycometabolic disturbance in patients with primary hypertension by declining insulin resistance.
Objective To investigate the heterotopic odontogenesis ability ofDelta1 gene transfected human dental pulp stem cell (DPSC) and nanohydroxyapatite/collagen (nHAC) composite scaffold. Methods The cultured human DPSC was transfected with Delta1-enhanced green fluorescent protein recombinant retrovirus supernatant，and was selected by puromycin to obtain the positive cell clone. The experimental group contained the Delta1 transfected DPSC; however, the control group did not contain the Delta1 transfected DPSC but contained DPSC transfected with vectors only. The cells were seeded into the nHAC carriers and were cultured in the odonto-inductive medium. The growth of the transduced cells in the carriers was observed by the fluorescent phase contrast microscope and the scanning electron microscope (SEM). The cell-carrier composites were subcutaneously transplanted into the Delta1 transfected 8 nude mice (female, 8 weeks old). Eight weeks after operation,the composites were taken out and tested with the histological and the immunohistological methods.Results Green fluorescence was observed inthe cells in the experimental group, which were grown in the carriers by the fluorescent phase contrast microscope. Observed by SEM, great amounts of transduced DPSC were observed along the scaffold materials, even filling the porous structures of nHAC and secreting a lot of extracellular matrix. However, in the control group, much fewer cells were found in the carriers. All the 4 Delta1 transduced DPSC-nHAC composites produced dentin-like structures that lined the surfaces of some nHAC porous structures. The odontoblast-like cells extended the cytoplasmic processes into the dentinal matrix, which was interfaced with a pulp-like interstitial tissue infiltrated with the blood vessels. Dentin sialophosphoprotein was expressed in the odontoblast-like cells when immunohisochemistry was performed. The morphology of the control composite was a typical one of the fibrous connective tissue,and only a little dentin-like structure was found in 2 of the 8 control transplants. Conclution DPSC can be used as the recipient cell of the Delta1 gene for expression and secretion of the Delta1 protein. The composites of the transfected cells and nHAC can induce heterotopic odontogenesis, which indicates that Delta1 is a novel candidate for the gene enhanced dentinpulp composite engineering.
Objective To investigate the clinical results of allograft and sural neurovascular flap in repairing calcaneus and skin defects.Methods From February 1996 to December 2002, allograft and sural neurovascular flap were used to repair calcaneusand skin defects in 6 cases. The causes included road accident in 3 cases, strangulation in 2 cases and crashing object in 1 case. The defect locations were at theback of the calcaneus( 1/3, 1/2 and 2/3 of calcaneus in 3 cases, 2 cases and 1case respectively). The flap area ranged from 6 cm×7 cm to 12 cm×17 cm. Results The flaps survived completely in 4 cases; the distal flaps necrosed partly in 2 cases and the wound healed by dressing. The postoperative X-ray films showed that the repaired bone and joint had normal position and the arcus plantaris recovered. After a follow upof 6 months to 3 years all the patients were achieved bone union in allograft and had no complications of absorption, infection and repulsion. The weightbearing and walking functions were restored and the injured foot obtained a satisfactory contour. After 36 months of operation, the sensory recovery of foot occurred. Conclusion The used-allograft iseasy to be obtained and arcus plantaris is easy to recover. The reversesural neurovascular- flap in repairing calcaneus and skin defects has the following advantages: the maintenance of blood supply for injured foot, the less dangerous operation, the simple procedure, the recovery of walking function, and the good appearance and sensation.