CN108117585A - A kind of target imports the polypeptide that siRNA promotes Apoptosis of Breast Cancer - Google Patents
A kind of target imports the polypeptide that siRNA promotes Apoptosis of Breast Cancer Download PDFInfo
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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Abstract
It the present invention relates to the technical field of biological control of breast cancer disease, and discloses a kind of target and imports the polypeptide that siRNA promotes Apoptosis of Breast Cancer, including polypeptide 1, the sequence of polypeptide 1 is：H Ile Phe D Trp Leu Leu Trp Gln Gly Arg Gly Gly Gly Arg Arg Arg Arg Arg Arg Arg OH, a kind of target import the polypeptide detection methods that siRNA promotes Apoptosis of Breast Cancer, comprise the following steps：1）Breast cancer cell culture：Breast cancer cell MDA MB 231 are chosen, and breast cancer cell MDA MB 231 are used into DMEM medium cultures, train the streptomysin for adding 10% hyclone, 100 units per ml penicillin and 100g/mL in above-mentioned foster base successively.The targeting imports the polypeptide that siRNA promotes Apoptosis of Breast Cancer, siRNA, which is wrapped up, using polypeptide 1 forms nano-particle come targeted delivery siRNA to breast cancer cell, nano-particle can not only pass through breast cancer cell surface receptor targeted delivery siRNA, and normal cell is damaged small, improve the practicability of polypeptide 1, cause the apoptosis of breast cancer cell using 1 targeted delivery TRPC1 siRNA of polypeptide simultaneously, so as to achieve the effect that treat breast cancer.
Description
Technical field
The present invention relates to the technical field of biological control of breast cancer disease, are specially that a kind of targeting imports siRNA promotion mammary gland
The polypeptide of cancer cell-apoptosis.
Background technology
At present, the treatment of breast cancer includes five bulks：It is operative treatment, chemotherapy, endocrine therapy, radiotherapy and molecule respectively
Targeted therapy.The development of molecular biology and to pathogenesis from the deep understanding of cell, molecular level so that oncotherapy into
The epoch of a new molecular targeted therapy are entered.Compared with chemotherapy, radiotherapy, molecular targeted therapy efficiently and selectively kills
Tumour cell so that treat more targeted and Small side effects.
At present, targeted delivery siRNA has very big potentiality in Field of Drug Discovery to specific cell, and siRNA not only may be used
To inhibit the expression of target gene as a research tool, while can also a kind of therapy be used as to treat various diseases.So
And siRNA targeted deliveries to breast cancer cell is very troublesome, existing substance tends to pair in targeted delivery siRNA
Normal cell damages, and influences the practicability of the technology, for this purpose, we devise a kind of targeting peptides.
The content of the invention
（One）The technical issues of solution
In view of the deficiencies of the prior art, the present invention provides it is a kind of target import siRNA promote Apoptosis of Breast Cancer polypeptide,
Solve the problems, such as that existing targeted delivery siRNA is inconvenient and easily normal cell is damaged.
（Two）Technical solution
To achieve the above object, the present invention provides following technical solution：A kind of targeting imports siRNA and promotes Apoptosis of Breast Cancer
Polypeptide, including polypeptide 1, the sequence of the polypeptide 1 is：
H-Ile-Phe-D-Trp-Leu-Leu-Trp-Gln-Gly-Arg-Gly-Gly-Gly-Arg-Arg-Arg-Arg-Arg-
Arg-Arg-OH。
A kind of target imports the polypeptide detection methods that siRNA promotes Apoptosis of Breast Cancer, comprises the following steps：
1）Breast cancer cell culture：Breast cancer cell MDA-MB-231 is chosen, and breast cancer cell MDA-MB-231 is used
DMEM medium cultures are trained in above-mentioned foster base and add 10% hyclone, 100 units per ml penicillin and 100g/mL successively
Streptomysin prepares breast cancer cell culture solution, and controlled at 37 DEG C, above-mentioned solution is placed on the titanium dioxide that concentration is 5%
When culture 24-72 is small in carbon incubator.
2）Polypeptide 1/siRNA binding abilities are tested：With various concentration than polypeptide 1 carry out gel blocking with siRNA respectively
Analysis test, prepares compound, and uses heparin（Go polyanion stable composite agent）Act on above-mentioned compound, observation knot
Fruit, and recorded.
3）Polypeptide 1/siRNA complex stabilities are tested：Take step 2）The polypeptide 1/siRNA compounds of middle preparation, will be upper
Addition RNA nucleases in compound are stated, and the mixture that above-mentioned compound is formed with RNA nucleases is placed on mice serum and incubates
It educates under environment, polypeptide 1/siRNA compounds is detected in the stability that RNA nucleases and mice serum are incubated under environment,
Simultaneously with the stability with gel retardation assay test siRNA after heparin release siRNA, test result is recorded.
4）Apoptosis of Breast Cancer experimental study：After being transfected by Westernblo detections with polypeptide 1/siRNA compounds
The band variation of apoptosis index bax, and compared with control group, observe the strengths and weaknesses of polypeptide 1/siRNA compounds, remembered
Record.
5）The outer silencing experiments of mammary tumor cells specific genosome：It is answered by Westernblot detections with polypeptide 1/siRNA
The band variation of TRPC1 after object transfects is closed, and compared with control group, observes the strengths and weaknesses of polypeptide 1/siRNA compounds, into
Row record.
6）Statistical procedures：By above-mentioned steps 2）、3）、4）With 5）The data of middle record are with mean+/-standard error table
Show and SigmaPlot12.5 softwares is used to carry out non-paired t test, P<Think that the difference between two groups has statistics when 0.05
Meaning.
（Three）Advantageous effect
Compared with prior art, the present invention provides a kind of polypeptide for targeting and importing siRNA and promoting Apoptosis of Breast Cancer, possess
Advantageous effect：The targeting imports the polypeptide that siRNA promotes Apoptosis of Breast Cancer, and wrapping up siRNA using polypeptide 1 forms nanometer
Particle carrys out targeted delivery siRNA to breast cancer cell, and nano-particle can not only be targeted by breast cancer cell surface receptor and passed
SiRNA is sent, and it is small to normal cell damage, the practicability of polypeptide 1 is improved, while utilizes 1 targeted delivery TRPC1 of polypeptide
SiRNA causes the apoptosis of breast cancer cell, so as to achieve the effect that treat breast cancer.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical solution in the embodiment of the present invention is clearly and completely retouched
It states, it is clear that described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.Based on the present invention
In embodiment, the every other implementation that those of ordinary skill in the art are obtained without making creative work
Example, belongs to the scope of protection of the invention.
The present invention provides following technical solution：A kind of target imports the polypeptide that siRNA promotes Apoptosis of Breast Cancer, including
Polypeptide 1, the sequence of the polypeptide 1 are：
H-Ile-Phe-D-Trp-Leu-Leu-Trp-Gln-Gly-Arg-Gly-Gly-Gly-Arg-Arg-Arg-Arg-Arg-
Arg-Arg-OH。
A kind of target imports the polypeptide detection methods that siRNA promotes Apoptosis of Breast Cancer, comprises the following steps：
1）Breast cancer cell culture：Breast cancer cell MDA-MB-231 is chosen, and breast cancer cell MDA-MB-231 is used
DMEM medium cultures are trained in above-mentioned foster base and add 10% hyclone, 100 units per ml penicillin and 100g/mL successively
Streptomysin prepares breast cancer cell culture solution, and controlled at 37 DEG C, above-mentioned solution is placed on the titanium dioxide that concentration is 5%
When culture 24-72 is small in carbon incubator.
2）Polypeptide 1/siRNA binding abilities are tested：With various concentration than polypeptide 1 carry out gel blocking with siRNA respectively
Analysis test, prepares compound, and uses heparin（Go polyanion stable composite agent）Act on above-mentioned compound, observation knot
Fruit, and recorded, when polypeptide 1 and siRNA ratios are more than 100:When 1, polypeptide 1 and siRNA binding abilities gradually enhance.
3）Polypeptide 1/siRNA complex stabilities are tested：Take step 2）The polypeptide 1/siRNA compounds of middle preparation, will be upper
Addition RNA nucleases in compound are stated, and the mixture that above-mentioned compound is formed with RNA nucleases is placed on mice serum and incubates
It educates under environment, polypeptide 1/siRNA compounds is detected in the stability that RNA nucleases and mice serum are incubated under environment,
Simultaneously with heparin discharge siRNA after with gel retardation assay test siRNA stability, test result is recorded, containing
The stability for having the more exposed siRNA of polypeptide 1/siRNA compounds in the solution of nuclease significantly improves, the polypeptide in mice serum
The stability of the more exposed siRNA of 1/siRNA compounds significantly improves.
4）Apoptosis of Breast Cancer experimental study：After being transfected by Westernblo detections with polypeptide 1/siRNA compounds
The band variation of apoptosis index bax, and compared with control group, observe the strengths and weaknesses of polypeptide 1/siRNA compounds, remembered
Record.
5）The outer silencing experiments of mammary tumor cells specific genosome：It is answered by Westernblot detections with polypeptide 1/siRNA
The band variation of TRPC1 after object transfects is closed, and compared with control group, observes the strengths and weaknesses of polypeptide 1/siRNA compounds, into
Row record, more normal group of polypeptide 1/TRPC1 siRNA compounds have the trend for promoting apoptosis, polypeptide 1/TRPC1 siRNA compounds
More normal group has the trend that TRPC1 is promoted to lower.
6）Statistical procedures：By above-mentioned steps 2）、3）、4）With 5）The data of middle record are with mean+/-standard error table
Show and SigmaPlot12.5 softwares is used to carry out non-paired t test, P<Think that the difference between two groups has statistics when 0.05
Meaning, while the above results demonstrate the feasibility that polypeptide 1 imports TRPC1 siRNA, are provided for siRNA treatments breast cancer latent
In new theoretical foundation and method.
The beneficial effects of the invention are as follows：SiRNA, which is wrapped up, using polypeptide 1 forms nano-particle come targeted delivery siRNA to breast
Adenocarcinoma cell, nano-particle can not only be damaged by breast cancer cell surface receptor targeted delivery siRNA, and to normal cell
It is bad small, the practicability of polypeptide 1 is improved, while causes withering for breast cancer cell using 1 targeted delivery TRPC1 siRNA of polypeptide
It dies, so as to achieve the effect that treat breast cancer, solves existing targeted delivery siRNA inconveniences and easily normal cell is made
Into the problem of destruction.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of variations, modification, replace
And modification, the scope of the present invention is defined by the appended.
Claims (2)
1. a kind of target imports the polypeptide that siRNA promotes Apoptosis of Breast Cancer, including polypeptide 1, which is characterized in that the polypeptide
1 sequence is：
H-Ile-Phe-D-Trp-Leu-Leu-Trp-Gln-Gly-Arg-Gly-Gly-Gly-Arg-Arg-Arg-Arg-Arg-
Arg-Arg-OH。
2. a kind of target imports the polypeptide detection methods that siRNA promotes Apoptosis of Breast Cancer, which is characterized in that including following step
Suddenly：
1）Breast cancer cell culture：Breast cancer cell MDA-MB-231 is chosen, and breast cancer cell MDA-MB-231 is used
DMEM medium cultures are trained in above-mentioned foster base and add 10% hyclone, 100 units per ml penicillin and 100g/mL successively
Streptomysin prepares breast cancer cell culture solution, and controlled at 37 DEG C, above-mentioned solution is placed on the titanium dioxide that concentration is 5%
When culture 24-72 is small in carbon incubator；
2）Polypeptide 1/siRNA binding abilities are tested：With various concentration than polypeptide 1 carry out gel retardation assay with siRNA respectively
Test, prepares compound, and uses heparin（Go polyanion stable composite agent）Act on above-mentioned compound, observation as a result,
And it is recorded；
3）Polypeptide 1/siRNA complex stabilities are tested：Take step 2）The polypeptide 1/siRNA compounds of middle preparation, will be above-mentioned multiple
Addition RNA nucleases in object are closed, and the mixture that above-mentioned compound and RNA nucleases are formed is placed on mice serum and is incubated ring
Under border, polypeptide 1/siRNA compounds are detected in the stability that RNA nucleases and mice serum are incubated under environment, simultaneously
With the stability with gel retardation assay test siRNA after heparin release siRNA, test result is recorded；
4）Apoptosis of Breast Cancer experimental study：Apoptosis after being transfected by Westernblo detections with polypeptide 1/siRNA compounds
The band variation of index bax, and compared with control group, observe the strengths and weaknesses of polypeptide 1/siRNA compounds, recorded；
5）The outer silencing experiments of mammary tumor cells specific genosome：Pass through Westernblot detections polypeptide 1/siRNA compounds
The band variation of TRPC1 after transfection, and compared with control group, observe the strengths and weaknesses of polypeptide 1/siRNA compounds, remembered
Record；
6）Statistical procedures：By above-mentioned steps 2）、3）、4）With 5）The data of middle record are represented simultaneously with mean+/-standard error
Non-paired t test, P are carried out with SigmaPlot12.5 softwares<Think that the difference between two groups is anticipated with statistics when 0.05
Justice.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113929752A (en) * | 2021-08-25 | 2022-01-14 | 天津大学 | Complex vesicle with double-way cancer cell inhibition effect, and preparation method and application thereof |
| WO2022206738A1 (en) | 2021-03-29 | 2022-10-06 | 南京大学 | Rna plasmid delivery system and application thereof |
| WO2022206739A1 (en) | 2021-03-29 | 2022-10-06 | 南京大学 | Viral vector-based rna delivery system and use thereof |
| WO2022206819A1 (en) | 2021-03-30 | 2022-10-06 | 南京大学 | Rna delivery system for treatment of huntington's disease |
| WO2022206788A1 (en) | 2021-03-29 | 2022-10-06 | 南京大学 | Nucleic acid delivery system and application thereof |
| WO2022206734A1 (en) | 2021-03-29 | 2022-10-06 | 南京大学 | Gene circuit, rna delivery system and use thereof |
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| CN103889457A (en) * | 2011-05-24 | 2014-06-25 | 波利威乐赞助有限公司 | Compositions and methods for efficacious and safe delivery of sirna using specific chitosan-based nanocomplexes |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022206738A1 (en) | 2021-03-29 | 2022-10-06 | 南京大学 | Rna plasmid delivery system and application thereof |
| WO2022206739A1 (en) | 2021-03-29 | 2022-10-06 | 南京大学 | Viral vector-based rna delivery system and use thereof |
| WO2022206788A1 (en) | 2021-03-29 | 2022-10-06 | 南京大学 | Nucleic acid delivery system and application thereof |
| WO2022206734A1 (en) | 2021-03-29 | 2022-10-06 | 南京大学 | Gene circuit, rna delivery system and use thereof |
| WO2022206819A1 (en) | 2021-03-30 | 2022-10-06 | 南京大学 | Rna delivery system for treatment of huntington's disease |
| CN113929752A (en) * | 2021-08-25 | 2022-01-14 | 天津大学 | Complex vesicle with double-way cancer cell inhibition effect, and preparation method and application thereof |
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