Animals ; Heart ; growth & development ; Myocardium ; metabolism ; Nerve Growth Factors ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

The neurotrophin receptor kinase (NTRK) gene encodes neurotrophic factor receptor tyrosine kinase (NTRK), which plays an important role in the development and function of the nervous system. NTRK gene fusion mutation results in the production of chimeric NTRK proteins, which have carcinogenic potential through constitutive activation or overexpression. NTRK gene fusion mutation can lead to a special type of wild type gastrointestinal stromal tumor (GIST), whose clinical manifestations and treatment are completely different from other types of GIST. This fusion mutation can be detected clinically by a variety of methods, including tumor DNA and RNA sequencing and immunohistochemical staining. In patients with NTRK fusion positive tumors, NTRK inhibitors such as larotrectinib and entrectinib have shown good antitumor efficacy, with clinical response rates as high as 75%. Therefore, there is a need to improve the recognition and detection of fuch patients and to improve their prognosis by individualized and precise treatment with TRK inhibitors.
Gastrointestinal Stromal Tumors/genetics* ; Gene Fusion ; Humans ; Neoplasms ; Nerve Growth Factors ; Protein Kinase Inhibitors ; Receptor, trkA/genetics* ; Receptors, Nerve Growth Factor/genetics*

Gene therapy is currently investigated in animal studies for treating erectile dysfunction (ED), and is affording an conspicuous therapeutic possibility for the treatment of ED, especially in L-arg-NO-cGMP pathway, ion channel, the protection of nerves and endothelia in erectile tissues. However there still exist so many problems for gene therapy to be effectively applied to the clinical treatment of ED. This review aims to examine the experimental efforts in recent years and tries to give a brief introduction to the new approaches in the field of ED researches.
Animals ; Cell Communication ; genetics ; Erectile Dysfunction ; therapy ; Genetic Therapy ; Ion Channels ; genetics ; Male ; Nerve Growth Factors ; genetics ; Rats ; Transfection ; Vascular Endothelial Growth Factor A ; genetics

Osteogenesis imperfecta (OI) comprises a heterogeneous group of disorders characterized by bone fragility, frequent fractures, and low bone mass. Dominantly inherited COL1A1 or COL1A2 mutations appear to be causative in the majority of OI types, but rare recessively inherited genes have also been reported. Recently, SERPINF1 has been reported as another causative gene in OI type VI. To date, only eight SERPINF1 mutations have been reported and all are homozygous. Our patient showed no abnormalities at birth, frequent fractures, osteopenia, and poor response on pamidronate therapy. At the time of her most recent evaluation, she was 8 yr old, and could not walk independently due to frequent lower-extremity fractures, resulting in severe deformity. No clinical signs were seen of hearing impairment, blue sclera, or dentinogenesis imperfecta. In this study, we describe the clinical and radiological findings of one Korean patient with novel compound heterozygous mutations (c.77dupC and c.421dupC) of SERPINF1.
Bone Density/genetics ; Child ; Collagen Type I/genetics ; Eye Proteins/*genetics ; Female ; Fractures, Bone/genetics ; Humans ; Nerve Growth Factors/*genetics ; Osteogenesis Imperfecta/diagnosis/*genetics ; Serpins/*genetics

To get the mature peptide of human-derived neurotrophin-6 (NT-6), NT-6 gene encoding mature peptide was amplified by PCR, using the NT-6 cDNA that had been cloned as templet. The gene encoding mature peptide of NT-6 gene was cloned into pGEX1-lambdaT plasmid to construct the fusion expression vector. Expression of fusion protein in Escherichia coli was defected after induction by isopropyl beta-D-thiogalactoside(IPTG). The mature peptide of NT-6 was collected with GST fusion protein purifying kit. It was shown that a fragment of 460bp was gained by PCR. With the techniques of double-cleave and electrophoresis, the recombinant vector was identified as pGEX1-NT-6. The recombinant vector pGEX1-NT-6 transformed Escherichia coli expressed fusion protein of 41KD after induction by IPTG. Cleaved by thrombin, the mature peptide of NT-6 was obtained; its molecular weight was about 15KD. The cloning and expression of human-derived NT-6 gene encoding mature protein has provided a basis for further studies on the function and clinical application of NT-6.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Isopropyl Thiogalactoside ; pharmacology ; Nerve Growth Factors ; biosynthesis ; genetics ; Peptides ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

BACKGROUNDThe mechanism of retinal neovascularization is not understood completely. Many growth factors are involved in the process of retinal neovascularization, such as vascular endothelial growth factor (VEGF) and pigment epithelium-deprived factor (PEDF), which are the representatives of angiogenic and antiangiogenic molecules respectively. Oxygen induced retinopathy (OIR) is a useful model to investigate retinal neovascularization. The present study was conducted to investigate the feasibility of small interference RNA (siRNA) targeting VEGF gene in attenuating oxygen induced retinopathy (OIR) by regulating VEGF to PEDF ratio (VEGF/PEDF).

METHODSIn vitro, cultured EOMA cells were transfected with VEGF-siRNA (psi-HI(TM)/EGFP/VEGF siRNA) and Lipofectamine(TM) 2000 for 24, 48, and 72 hours, respectively. Expression of VEGF mRNA was evaluated by real time polymerase chain reaction (PCR) and the level of VEGF protein was analyzed by Western blotting. In vivo, OIR model mice were established, the mice (C57BL/6J) received an intra-vitreal injection of 1 µl of mixture of psi-HI(TM)/EGFP/VEGF siRNA and Lipofectamine 2000. Expressions of retinal VEGF and PEDF protein were measured by Western blotting, retinal neovascularization was observed by fluorescein angiography, and quantified.

RESULTSIn vitro psi-HI(TM)/EGFP/VEGF siRNA treatment significantly reduced VEGF mRNA and protein expression. In vivo, with decreased VEGF and VEGF-PEDF ratio, significant attenuation of neovascular tufts, avascular regions, tortuous, and dilated blood vessels were observed in the interfered animals.

CONCLUSIONSVEGF plays an important role in OIR, and the transfection of VEGF-siRNA can effectively downregulate VEGF expression in vivo, accompanied by the downregulation of VEGF-PEDF ratio, and simultaneous attenuation of retinal neovascularization was also observed. These findings suggest that VEGF/PEDF may serve as a potential target in the treatment of retinal neovascularization and RNA interference targeting VEGF expression, which represents a possible therapeutic strategy.

Animals ; Eye Proteins ; analysis ; Mice ; Mice, Inbred C57BL ; Nerve Growth Factors ; analysis ; RNA, Small Interfering ; genetics ; Retinal Neovascularization ; therapy ; Serpins ; analysis ; Vascular Endothelial Growth Factor A ; analysis ; genetics ; physiology

OBJECTIVETo explore the protective mechanism of penetration therapy with scalp electroacupuncture on Parkinson's disease (PD).

METHODSThirty-six Wistar rats were randomly divided into a normal group, a sham operation group, a model group and a penetration therapy group, 9 rats in each group. The sham operation group was operated by micro-injection with normal saline in the left corpus striatum. The model group and penetration therapy group were generated by micro-injection with 6-hydroxydopamine in the left corpus striatum to prepare rotation model of PD. The penetration therapy group was treated by penetration therapy with scalp electroacupuncture through "Baihui" (GV 20) to "Taiyang" (EX-HN 5), once each day, 6 days for one course, altogether 2 courses, and there was no treatment in the other two groups. (1) Immunohistochemical method was used to test the morphology and count of positive cells of tyrosine hydroxylase (TH). (2) In situ hybridization histochemistry was applied to detect the mRNA expression of brain-derived neurotrophic factor (BDNF).

RESULTS(1) The areal density (AD), numerical density (ND) and integrating optic density (IOD) of the positive neurons of TH in substantia nigra in the penetration therapy group were 0.065 +/- 0.011, 0.014 +/- 0.003 and 0.470 +/- 0.099, respectively, which were higher than 0.039 +/- 0.008, 0.008 +/- 0.002 and 0.266 +/- 0.065 in the model group (all P < 0.05). (2) The AD, ND and IOD of the mRNA expression of BDNF in substantia nigra in the penetration therapy group were 0.100 +/- 0.012, 0.014 +/- 0.003 and 1.158 +/- 0.130, respectively, which were higher than 0.047 +/- 0.012, 0.007 +/- 0.001 and 0.602 +/- 0.108 in the model group (all P < 0.05).

CONCLUSIONThe penetration therapy with scalp electroacupuncture has a better protective effect on dopaminergic neurons in substantia nigra of rats with PD. The protective mechanism is related to the provocation of neural nourishment so as to improve the morphous of dopaminergic neurons and increase the number of dopaminergic neurons.

Animals ; Disease Models, Animal ; Electroacupuncture ; Female ; Gene Expression ; Humans ; Male ; Nerve Growth Factors ; genetics ; metabolism ; Parkinson Disease ; genetics ; metabolism ; therapy ; Rats ; Rats, Wistar ; Scalp ; Substantia Nigra ; metabolism

Vectors used to carry foreign genes play an important role in gene therapy, among which, the adeno-associated virus (AAV) has many advantages, such as nonpathogenicity, low immunogenicity, stable and long-term expression and multiple-tissue-type infection, etc. These advantages have made AAV one of the most potential vectors in gene therapy, and widely used in many clinical researches, for example, Parkinson's disease. This paper introduces the biological characteristics of AAV and the latest research progress of AAV carrying neurotrophic factor, dopamine synthesis related enzymes and glutamic acid decarboxylase gene in the gene therapy of Parkinson's disease.
Animals ; Aromatic-L-Amino-Acid Decarboxylases ; genetics ; Dependovirus ; genetics ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; Glutamate Decarboxylase ; genetics ; Humans ; Nerve Growth Factors ; genetics ; Neurturin ; genetics ; Parkinson Disease ; therapy

OBJECTIVETo identify genes expressed in the fetal heart that are potentially important for myocardial development and cardiomyocyte proliferation.

METHODSmRNAs from fetal (29 weeks) and adult cardiomyocytes were use for suppression subtractive hybridization (SSH). Both forward (fetal as tester) and reverse (adult as driver) subtractions were performed. Clones confirmed by dot-blot analysis to be differentially expressed were sequenced and analyzed.

RESULTSDifferential expressions were detected for 39 out of 96 (41%) clones on forward subtraction and 24 out of 80 (30%) clones on reverse. For fetal dominating genes, 28 clones matched to 10 known genes (COL1A2, COL3A1, endomucin, HBG1, HBG2, PCBP2, LOC51144, TGFBI, vinculin and PND), 9 clones to 5 cDNAs of unknown functions (accession AK021715, AF085867, AB040948, AB051460 and AB051512) and 2 clones had homology to hEST sequences. For the reverse subtraction, all clones showed homology to mitochondrial transcripts.

CONCLUSIONSWe successfully applied SSH to detect those genes differentially expressed in fetal cardiac myocytes, some of which have not been shown relative to myocardial development.

Aged ; Cells, Cultured ; Collagen ; Collagen Type I ; Collagen Type III ; genetics ; DNA-Binding Proteins ; genetics ; Forkhead Transcription Factors ; Gene Expression ; physiology ; Heart ; embryology ; growth & development ; Heterogeneous-Nuclear Ribonucleoproteins ; genetics ; Humans ; Nerve Tissue Proteins ; genetics ; Nucleic Acid Hybridization ; RNA-Binding Proteins ; Transcription Factors ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1 ; Vinculin ; genetics

Animals ; Food Hypersensitivity ; genetics ; immunology ; metabolism ; Forkhead Transcription Factors ; genetics ; metabolism ; Glucocorticoid-Induced TNFR-Related Protein ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; genetics ; Receptors, Nerve Growth Factor ; genetics ; metabolism ; Receptors, Tumor Necrosis Factor ; genetics ; metabolism ; T-Lymphocytes, Regulatory ; metabolism