Application of LamG3/4/5 of laminin alpha2 functional region in preparation of medicine for preventing contraceptive induced testis injury

A technology of laminin and functional area, applied in the application field of medicine, can solve the problem of no spermatogenic epithelial damage and the like

A technology of laminin and functional area, applied in the application field of medicine, can solve the problem of no spermatogenic epithelial damage and the like

CN112516293APending Publication Date: 2021-03-19THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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  • Application of LamG3/4/5 of laminin alpha2 functional region in preparation of medicine for preventing contraceptive induced testis injury
  • Application of LamG3/4/5 of laminin alpha2 functional region in preparation of medicine for preventing contraceptive induced testis injury
  • Application of LamG3/4/5 of laminin alpha2 functional region in preparation of medicine for preventing contraceptive induced testis injury

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Cloning method of LamG3 / 4 / 5 coding gene fragment

[0032] Using SD rat testis cDNA, design cloning primers with reference to the rat laminin α2 coding gene sequence: upstream primer: CCGCTCGAGATGGTCGGAACGGAAATCAACCTG (SEQ ID NO.3);

[0033] Downstream primer: ACGCGTCGACTTACAGAGCCTTGGCAAAATTAAC (SEQ ID NO.4);

[0034]引物中包括限制性内切酶位点XhoI / SalI、启动 / 中止密码子和保护性碱基,PCR扩增后得到LamG3 / 4 / 5编码基因片段,序列如SEQ ID NO.2所示,(GTCGGAACGGAAATCAACCTGTCCTTCAGTACCAGGAACGAGTCTGGGATCATTCTCTTGGGAAGTGGAGGGACACTCACACCACCCAGGAGAAAACGGAGACAAACTACACAGGCTTATTATGCCATATTCCTCAACAAGGGCCGTCTGGAAGTGCATCTCTCCTCGGGGACACGAACGATGAGGAAAATTGTCATCAAACCCGAGCCAAATTTGTTTCATGATGGGAGAGAACATTCTGTCCACGTAGAAAGAACCAGAGGAGTCTTCACTGTTCAAGTTGATGAAAACAGAAGACATATGCAAAACCTGACAGAGGAGCAGCCCATCGAAGTGAAAAAGCTCTTTGTTGGGGGTGCTCCTCCTGAATTCCAGCCCTCCCCACTCAGAAATATCCCAGCTTTTCAAGGCTGTGTGTGGAACCTTGTTATTAACTCCATCCCCATGGACTTTGCACAGCCTATAGCTTTCAAAAATGCTGATATTGGCCGCTGTGCCTATCAAAAGCCCCGGGAAGAAGAAGACGATGCCGTTCCAGCTGAAGTTACTGTCCAGCCTCAGCCAGTGCCTACCCCTGCCTTCCCTTT...

Embodiment 2

[0036] 1. Sequencing of LamG3 / 4 / 5 coding gene fragments

[0037] The PCR product of the coding gene fragment obtained in Example 1 was recovered and sent to GeneWiz Company for sequencing. The sequencing results showed that the homology with the reference rat laminin α2 coding gene sequence XM_017590489.1 was more than 99%, indicating that LamG3 / 4 / 5 was successfully cloned coded gene segment.

[0038] 2. Construction of LG3 / 4 / 5 fragment expression vector

[0039] The cloned LamG3 / 4 / 5 coding gene fragment was connected to the eukaryotic expression vector pCI-neo (purchased from Promega) at the XhoI / SalI multiple cloning site to obtain the expression vector pCI-neo-LG3 / 4 / 5. XhoI / SalI double enzyme digestion was performed on the obtained recombinant expression vector. Restriction map see figure 2 , figure 2 Electropherogram of the digested product after double digestion of the constructed pCI-neo-LG3 / 4 / 5 overexpression vector. Depend on figure 2 It can be seen that the l...

Embodiment 3

[0041] Method for studying the preventive effect of pCI-neo-LG3 / 4 / 5 overexpression in rat testis on oral Adjudin-induced testicular injury

[0042] Grouping method: A total of 18 male SD rats were divided into 3 groups with 6 rats in each group. Among them, 6 mice in pCI-neo empty plasmid transfection group (blank control group), 6 mice treated with Adjudin (positive control group), and 6 mice in Adjudin group after transfection with pCI-neo-LamG3 / 4 / 5 overexpression vector (test group).

[0043] Experimental method: For the experimental group and the blank control group, the recombinant expression vector pCI-neo-LamG3 / 4 / 5 or the empty plasmid pCI-neo was transfected into the testis of SD rats, and each testis was injected with 15 μg of the recombinant expression vector (each testis The total injection volume is 70 μl, which contains 15 μg of plasmid DNA, 1.8 μl of in-vivojet PEI transfection reagent, dissolved in 5% sterile glucose solution), and the testis is injected three ...

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Abstract

The invention relates to application of LamG3 / 4 / 5 of a laminin alpha2 functional region in preparation of a medicine for preventing male contraceptive induced testis injury, and belongs to the technical field of medicine preparation. An amino acid sequence of the LamG3 / 4 / 5 of the laminin alpha2 functional region is shown as SEQ ID NO. 1. Overexpression drugs of LamG3 / 4 / 5 of the laminin alpha 2 functional region can effectively prevent testis injury induced by male contraceptives, and can play an obvious role in preventing structural integrity damage of seminiferous epithelium of seminiferous tubules in a testis, lesion of the seminiferous tubules and cytoskeletal protein damage / destruction of the seminiferous epithelium of the seminiferous tubules in the testis which are caused by oral administration of Adjudin.

Description

technical field [0001] The invention relates to the technical field of medicine preparation, in particular to the application of laminin α2 functional region LamG3 / 4 / 5 in the preparation of medicines for preventing testicular damage induced by male contraceptives. Background technique [0002] In male mammals, the testicular seminiferous tubule is the starting point of spermatogenesis, in which Sertoli cells, located on the basement membrane of the seminiferous tubule, extend into the lumen, and branch into the germ cells of different stages. One of the important functions of Sertoli cells is to constitute one of the tightest blood-tissue barrier structures in the body, namely the blood-testis barrier. The functions of the blood-testis barrier include preventing cytotoxic substances from entering the lumen of the seminiferous tubules and preventing autoimmune reactions induced by sperm. Adjudin (aka AF2364) is an indazolyl compound with reversible non-hormonal contraceptive...

Claims

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Application Information

Patent Timeline
19 Mar 2021
Publication
CN112516293A
IPC
A61K38/39; C07K14/78; C12N15/12; C12N15/63; A61P15/00; A61P15/08
CPC
A61K38/39; C07K14/78; A61P15/00; A61P15/08
Inventors
李林溪; 郑泉恩