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Small molecule polypeptide and application thereof in preparing medicine for preventing and treating ischemic stroke

A technology for ischemic stroke and small molecule peptides, applied in the field of medicine, can solve problems such as inappropriateness, and achieve the effects of no toxic side effects, high synthetic purity, and good solubility

Active Publication Date: 2019-04-02
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the important physiological role of glutamate receptors in normal neuronal activity, direct blockade of glutamate receptors is not suitable for the treatment of ischemic stroke

Method used

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  • Small molecule polypeptide and application thereof in preparing medicine for preventing and treating ischemic stroke
  • Small molecule polypeptide and application thereof in preparing medicine for preventing and treating ischemic stroke
  • Small molecule polypeptide and application thereof in preparing medicine for preventing and treating ischemic stroke

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Synthesis of TAT-CDK5-CTM

[0029] The sequence of TAT-CDK5-CTM is shown as SEQ ID NO.1, which was artificially synthesized by Jiangsu Qiangyao Biotechnology Co., Ltd. The synthesis report is as follows, and the chromatogram is as follows figure 1 shown.

[0030] TAT-CDK5-CTM synthetic HPLC report

[0031]

[0032] Table 1

[0033]

wavelength

time

Peak area

Peak Area Percentage (%)

1

220nm

7.310

17438

0.2265

2

220nm

9.067

55117

0.716

3

220nm

9.360

7404161

96.18

4

220nm

9.933

118775

1.543

5

220nm

11.207

38910

0.5055

6

220nm

12.239

63670

0.8271

[0034] The purity of the synthesized TAT-CDK5-CTM polypeptide is 96.18%, 1 mg per tube, it is white powder, completely soluble in water, sealed and protected from light, and stored at -20°C. Before use, dilute it with normal saline for injection according to...

Embodiment 2

[0038] TAT-CDK5-CTM blocks the binding of neuronal NR2B receptors to CDK5 under ischemia-hypoxic conditions, and inhibits neuronal apoptosis and necrosis caused downstream of CDK5.

[0039] The primary neurons on day 10 were cultured in vitro and treated with oxygen glucose deprivation (OGD) to mimic the cell model of ischemic stroke. After 90 minutes of OGD treatment, normal culture was performed for 2 hours, and 5uM TAT-CDK5-CTM polypeptide or control TAT-s-CDK5 polypeptide or vehicle was incubated. Cellular proteins were extracted after 2 hours. Anti-NR2B antibody was used to precipitate cellular protein, and then anti-CDK5 antibody was used to detect the precipitated protein. The black blot detected on the NC membrane indicated the interaction between NR2B and CDK5. The results showed that after administration of TAT-CDK5-CTM, the anti-CDK5 antibody could hardly detect the CDK5 protein on the NC membrane, suggesting that TAT-CDK5-CTM blocked the interaction between NR2B a...

Embodiment 3

[0041] Application of TAT-CDK5-CTM in cell model of ischemic stroke

[0042] (1) Construction of primary neuron culture and glucose and oxygen deprivation simulated ischemic stroke cell model

[0043] Fetal mice on the 18.5th day of the embryonic period were taken out from the uterus of pregnant mice, and the prefrontal cortex of the brain was taken out after decapitation in the dissection solution (Hank's Balance Solution), and the cells were digested with 0.125% trypsin. Cells were planted on 20ng / ul poly-lysine and laminin-coated coverslips, and the coverslips were placed in 12-well plates with a cell planting density of 100-150 cells per square millimeter and medium without Serum Neurobasal medium (21103, gibco) was added with 2% B27, and the same medium was used for half replacement every three days, that is, half of the old medium was discarded and half of the fresh medium was added. Immunofluorescent labeling of the cultured cells with the neuron marker β-tubulin III (...

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Abstract

The invention discloses a small molecule polypeptide and an application thereof in preparing a medicine for preventing and treating ischemic stroke. By synthesizing a TAT protein transduction domain,the protein polypeptide of CDK5 and the fusion protein TAT-CDK5-CTM of the autophagy targeted motif CTM can be combined and inhibited, the TAT is used to carry the CDK5 protein polypeptide, which is transported by blood through a blood cerebral barrier, and is taken up by neurons. The peptide is applied to in-vitro and in-vivo ischemic stroke models to effectively block the bonding effect of the NR2B subunit of a N-methyl aspartate receptor (NMDAR) and the cyclin-dependent protein kinase 5 (CDK5), the CTM mediates the polypeptide molecules which are bonded with CDK5 protein to enter lysosomalfor degradation, neuronal apoptosis and necrosis induced by CDK5 downstream can be inhibited, brain damage after ischemic stroke can be reduced, and the application provides a molecular target for thedevelopment of drugs for the treatment of ischemic stroke.

Description

technical field [0001] The invention belongs to the field of medicine, relates to small molecular polypeptides and their medical applications, in particular to an artificially synthesized small molecular polypeptide TAT-CDK5-CTM, and the use of small molecular polypeptide TAT-CDK5-CTM in the preparation of prevention or treatment of ischemic stroke application in medicine. Background technique [0002] Ischemic stroke is a severe neurological disease in which the cerebral blood flow is interrupted, resulting in thrombosis or embolism, leading to cerebral ischemia. Patients will suffer from paralysis, language barriers, vision loss and even death. Because of its high morbidity, high mortality and high disability rate, it is considered as a common refractory disease that seriously endangers human health and life safety. According to the 2016 stroke epidemiology report, there are 70 million stroke patients in my country, 2 million new strokes every year, and 1.65 million strok...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K38/10A61P9/10A61P25/00
CPCA61K38/00C07K7/08C07K2319/10
Inventor 朱铃强周雅帆刘丹汪晶邓曼菲
Owner HUAZHONG UNIV OF SCI & TECH
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