CN104548138A

CN104548138A - Application of gene NOVA1 in preparation of medicine for treating nerve cell hypoxia injury

Info

Publication number: CN104548138A
Application number: CN201510044038.3A
Authority: CN
Inventors: 李华玲; 孔玲; 吕贝; 夏靖; 陈欣虹
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2015-01-29
Filing date: 2015-01-29
Publication date: 2015-04-29

Abstract

The invention relates to the field of medical biochemistry and in particular relates to a medicine for treating nerve cell hypoxia injury. A Nova1 hypoxia injury protective effect is explored by constructing a Nova1 eukaryotic expression vector and simulating neuron cell hypoxia conditions. A stable physical hypoxia injury model is built by taking PC12 cells, namely rat adrenal pheochromocytoma, as an object; the Nova1 eukaryotic expression vector Pcmv-myc-Nova1 is constructed by adopting digestion and PCR methods, and sequencing verification is carried out; and a cell transfection experiment is carried out, cellular morphology, RNA and protein level are detected, detection results find that a nerve specificity splicing factor Nova1 has a hypoxia injury protective effect, and a foundation is laid for research of a hypoxia injury protection mechanism in future.

Description

gene nOVA1 application in preparation treatment neurocyte anoxia-induced apoptosis medicine

Technical field

The present invention relates to Medical Biochemistry field, be specifically related to the medicine for the treatment of neurocyte anoxia-induced apoptosis.

Background technology

Brain is the most active organ of energy i (in vivo) metabolism, oxygen consumption blood flow and maximum, cerebral apoplexy has become that the world today is lethal, one of the principal disease that disables, but its mechanism is illustrated so far not yet completely, the main damage from energy exhaustion, oxygen free radical injury, lipid peroxidation, inflammatory cytokine and mediated by nitric oxide in the past is angularly studied; In treatment, adopt scavenging free radicals, alleviate the measure such as calcium overload, cytoprotective application, but general curative effect is very micro-.Brain as organ the most complicated in body, wherein the splicing factor of nerve-specific and regulation and control thereof obviously and the physiological status of brain and pathological state closely related.Nova1 is commonly used to as nerve-specific splicing factor the network studying its alternative splicing regulation and control, and the research in anoxia has no report.

Summary of the invention

The present invention, by building the carrier for expression of eukaryon of Nova1, the condition of imictron cell hypoxia, explores the anoxia-induced apoptosis protective effect of Nova1.

The present invention is with PC12 cell, and namely Clonal Rat Pheochromocytoma tumor is object, sets up stable physics hypoxia model: anoxia cell, oxygen concentration 0.1%;

Construct the carrier for expression of eukaryon of Nova1 by enzyme action and PCR method, Pcmv-myc-Nova1, and carry out sequence verification;

Carry out cell transfection assays, detect from cellular morphology, RNA and protein level, for the anoxia-induced apoptosis protective effect studying Nova1 is further laid a good foundation.

Beneficial effect:

The PC12 of the present invention by cultivating; namely PC12 cells is the model that representative establishes neurocyte anoxia-induced apoptosis; optimum culture condition; construct the carrier for expression of eukaryon pcmv-myc-Nova1 of nerve-specific splicing factor Nova1; transfect neuronal cells; detect from cellular morphology, RNA and protein level; and carried out the monitoring of different time points; find when anoxia 4h, 6h, 48h; Nova1 has protective effect to neurocyte, and this has had new prompting to the research of anoxia-induced apoptosis.Find that nerve-specific splicing factor Nova1 has the protective effect of anoxia-induced apoptosis, laying a good foundation for studying anoxia-induced apoptosis protection mechanism from now on, meanwhile, cerebral apoplexy can be studied further as the drug candidate of antisense oligonucleotide.

Accompanying drawing explanation

Fig. 1 PCR increases nova1.

Fig. 2 enzyme action qualification recombiant plasmid pcmv-myc-Nova1.

Fig. 3 Realtime-PCR detects each group of Nova1 mrna expression.

Fig. 4 take GAPDH as internal reference albumen, and the detection of WesternBlot method turns pCMV-Myc-Nova1 group and occurs specific protein band.

Fig. 5 WesternBlot detects each group of NOVA1 protein expression.

Fig. 6 MTT detects cell survival rate after each group of anoxia different time.

After Fig. 7 Realtime-PCR detects each group of anoxia 4h, 6h and 48h, Nova1 mRNA expresses.

Fig. 8 Western Blot detects NOVA1 protein expression after each group of anoxia 4h, 6h and 48h.

Detailed description of the invention

Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment does not limit in any form to the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.

Unless stated otherwise, embodiment of the present invention used medium and experimental condition are this area conventional medium and test.Unless stated otherwise, embodiment of the present invention agents useful for same is commercial.

embodiment 1the structure of the carrier for expression of eukaryon of Nova-1

We have purchased gene Homo sapiens neuro-oncological ventral antigen 1 (NOVA1) of Changsha Ying Run biotech company, transcript variant 1 NM_002515 BC075038, using cloning vehicle pCR4-TOPO as template, carry out PCR amplification, see that Fig. 1 PCR amplification nova1 obtains band clearly, obtain the Nova1 of 1500bp, (1,3, DL2000 Marker; 2,15 μ l pcr products; 4,7.5 μ l pcr products).Then carry out glue recovery, with sal I and xho I double digestion, glue is connected with the carrier for expression of eukaryon pcmv-myc through sal I and xho I double digestion after reclaiming again, proceeds to DH5 α and screens.And carry out enzyme action qualification recombiant plasmid, see that Fig. 2 enzyme action qualification recombiant plasmid pcmv-myc-Nova1:1 hole is DL2000 Marker, 2 holes are pcmv-myc-Nova1 digested by sal I and xho I, 3 holes is pcmv-myc digested by sal I and xho I.By sequence comparison on NCBI after order-checking, carried out forward and backward sequencing comparison, result proves successfully to be built into the carrier for expression of eukaryon of Nova-1.

embodiment 2carrier for expression of eukaryon pCMV-Myc-Nova1 transfection PC12 test cell line.

(1) Lipo-2000 transfection PC12 cytotoxicity detects.

With Lipo-2000 transfection PC12 cell, form Lipo-2000 transfection group, separately establish PC12 cell untransfected group as reference.

By untransfected group, Lipo-2000 transfection group, often organize 5 holes, detect different time cell survival rate with mtt assay, repeat 3 times, testing result shows, and Lipo-2000 transfection group is after transfection 24h, 48h, 72h and 96h, and cell survival rate comparatively untransfected group there is no obvious decline.

(2) carrier pCMV-Myc-Nova1 transfection PC12 cell.

With pCMV-Myc-Nova1 transfection PC12 cell formed turn pCMV-Myc-Nova1 group, separately establish untransfected group, and turn Pcmv-myc carrier, containing nova1 turn empty carrier group as reference.

(1) screening of transfection Best Times.

Collecting cell after 24h, 48h, 72h and 96h is cultivated respectively after pCMV-Myc-Nova1 being proceeded to PC12 cell, Realtime-PCR and WesternBlot method is adopted to detect: the RNA of extracting PC12 cell, measure value and the concentration of RNA OD260/280 with ultraviolet spectrophotometer, the value of RNA OD260/280 should between 1.8-2.0; Then reverse transcription is that cDNA does quantitative fluorescent PCR, in whole PCR process, only there is a specific peak in solubility curve, this shows to generate without primer dimer and nonspecific products, design of primers is reasonable, and whole process does not have contamination phenomenon yet, reference gene and the equal Absorbable organic halogens of genes of interest are expressed.

(2) expression after RT-PCR detection Nova1 gene transfection.

After carrier for expression of eukaryon pCMV-Myc-Nova1 transfection PC12 cell 72h, extracting RNA, carry out Realtime-PCR detection, result display is compared with untransfected group, the expression turning pCMV-Myc-Nova1 group Nova1 mRNA obviously raises, and has significant difference, sees Fig. 3, * compared with untransfected group, P < 0.05.This shows that pCMV-Myc-Nova1 successfully proceeds in PC12 cell, and successful expression Nova1 mRNA.And untransfected group, to turn empty carrier group Nova1 mrna expression amount extremely low.

(3) expression after WesternBlot detection NOVA1 gene transfection.

Albumen is extracted after cell transfecting 72h, detecting untransfected group by WesternBlot method, turn empty carrier group and turn the expression of pCMV-Myc-Nova1 group NOVA1 albumen, take GAPDH as internal reference albumen, shown in testing result, turn pCMV-Myc-Nova1 group and occur specific protein band, see Fig. 4, compared with untransfected group, expression obviously raises, there is significant difference, see Fig. 5, * compared with untransfected group, P < 0.05.This shows that pCMV-Myc-Nova1 successfully proceeds in PC12 cell further, and successful expression NOVA1 albumen, and untransfected group, to turn empty carrier group NOVA1 expressing quantity extremely low.

(4) Nova1 is to the research of PC12 cell hypoxia injury protection.

MTT detects each group of anoxia different time: cell survival rate after 0h 2h 4h 6h 8h 10h 12h 24h 48h 72h.

With mtt assay, cell survival rate detection is carried out to each group of cell, result shows: compared with the matched group of respective time point, after proceeding to pCMV-Myc-Nova1, anoxia 4h, 6h and 48h group cell survival rate obviously rise, significant difference, see Fig. 6, * compare with the matched group of respective time point, P < 0.05, and other each group differences are not remarkable.

Experimental result shows, Nova1 has the protective effect of anoxia-induced apoptosis, lays a good foundation for studying anoxia-induced apoptosis protection mechanism from now on.

The expression of each group cell Nova1 mRNA after detecting untransfected group by Realtime-PCR method, turn empty carrier group and turn pCMV-Myc-Nova1 group difference anoxia 4h, 6h, 48h, result shows: compare with respective untransfected group, anoxia 4h, 6h, 48h, turn pCMV-Myc-Nova1 group Nova1 mrna expression amount all obviously to raise, and significant difference, see Fig. 7, * compares with the untransfected group of respective time point, P < 0.05.

The expression of each group cell NOVA1 albumen after detecting untransfected group by Western Blot method, turn empty carrier group and turn pCMV-Myc-Nova1 group difference anoxia 4h, 6h, 48h, result shows: compare with respective untransfected group, three time points, turn pCMV-Myc-Nova1 group NOVA1 expressing quantity all obviously to raise, and significant difference, see Fig. 8, * compares with the untransfected group of respective time point, P < 0.05.

Claims

1. the application of gene NOVA1 in preparation treatment neurocyte anoxia-induced apoptosis medicine.