CN111317743B - Application of recombinant adeno-associated virus based on Kif11 gene in inhibition of pathological pain - Google Patents
Application of recombinant adeno-associated virus based on Kif11 gene in inhibition of pathological pain Download PDFInfo
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- CN111317743B CN111317743B CN202010177819.0A CN202010177819A CN111317743B CN 111317743 B CN111317743 B CN 111317743B CN 202010177819 A CN202010177819 A CN 202010177819A CN 111317743 B CN111317743 B CN 111317743B
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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Abstract
The invention discloses application of a recombinant adeno-associated virus based on a Kif11 gene in inhibiting pathological pain, belongs to the technical field of biological pharmacy, and discloses a recombinant adeno-associated virus rAAV9-ZsGreen-ShRNA-Kif11 containing a rat Kif11 gene interference shRNA sequence, which has the function of transfecting dorsal root ganglion nerve cells cultured in vitro; has the function of transfecting the nerve cells of the dorsal root ganglion of the living animal; has the function of inhibiting pathological pain sensitivity reaction, so the Kif11 gene has reference significance in pain treatment by taking the intervention target spot, and has application prospect in pain treatment medicines.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to application of recombinant adeno-associated virus based on a Kif11 gene in inhibiting pathological pain, in particular to application of Kif11 gene interference shRNA recombinant adeno-associated virus transfected nerve cells and inhibition of pathological pain thereof.
Background
Pathological pain is one of the important problems affecting human health at present, and the generation mechanism thereof is complex. Under the inducement effects of acute inflammation, tissue injury, nerve compression, tumor, chronic infection and the like, the excitability of nerve cells is abnormally increased, and the continuous progress of the pathophysiological processes can cause the structural and functional plasticity of the nervous system to be changed, so that the pain is continuously chronically changed and enters a disease state. There is currently no satisfactory therapeutic agent for pathological pain, especially chronic pain. The Dorsal Root Ganglion (DRG) neurons express a plurality of pain regulating molecules such as neurotransmitters, receptors and ion channels, are the first-order neurons for generating and transmitting pathological pain signals, and are important targets for exerting the functions of analgesic drugs. Several compounds have been developed for these pain modulating molecules, some of which have even been approved by the FDA in the united states for phase III clinical trials, but the therapeutic effect is not ideal and the best non-opioid analgesic drugs also inhibit the pain score by as much as 30%. Opioid analgesics are mainly used for cancer pain due to side effects such as addiction. Therefore, the problems of poor curative effect, poor specificity, multiple side effects and the like are bottlenecks which plague the research and development of the current analgesic drugs.
In recent years, the rapid development of RNA interference (RNAi) technology can specifically eliminate or close the expression of specific genes, and open up a new approach for the treatment of diseases. One of the main methods for realizing RNAi artificially at present is to directly introduce siRNA sequences synthesized in vitro into cells, and the main disadvantages are low transfection efficiency, unstable effect and short action duration. The other is continuous and stable expression of siRNA through transfection vector, the main advantage is that a large amount of short hairpin RNA (shRNA) is stably expressed, the ends of the shRNA are processed and treated in vivo to form siRNA-like molecules of about 21bp, and the siRNA can start RNAi so as to be used for long-time interference of the expression of target genes. Compared with retrovirus and lentivirus vectors, the adenovirus-associated virus (AAV) vector has the outstanding advantages of high safety, low immunogenicity, wide host range, long-term stable expression of the carried transfection vector gene, application in vitro and in vivo, and wide application range and prospect. Recent reports show that the RNAi technology is used for specifically interfering ion channels (such as potassium ion channels, sodium ion channels, glutamate receptors and the like) of nerve cells to play an analgesic role, and the RNA interference technology has potential in the aspect of research and development of pain drugs.
Kif11 (kinesin family member 11, also known as Eg 5) is one of the kinesin family members. Most researches suggest that the molecule is mainly involved in the positioning of chromosomes, the separation of centrosomes and the formation and separation of bipolar spindles in cell division, and therefore, the molecule can be used as a target of antitumor drugs. In recent years, it has been found that Kif11 is expressed in non-dividing nerve cells and is involved in regulating the growth of neurites, but it has not been clarified whether this molecule exerts other effects in nerve cells.
Although small interfering RNAs against Kif11 are currently available, such as siRNA, shRNA plasmids and lentiviral packaging particles produced by Santa Cruis, targeting the mouse Kif11 gene, the viral titer is low (1X 10 per ml) 6 Lentivirus transduction particles), whether nerve cells can be transfected and biological effects can be achieved, and the like are not reported.
Disclosure of Invention
In order to overcome the disadvantages of the prior art, the present invention aims to provide the application of recombinant adeno-associated virus based on Kif11 gene in inhibiting pathological pain. In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses application of recombinant adeno-associated virus rAAV9-ZsGreen-ShRNA-Kif11 based on Kif11 gene in preparation of an anti-pain medicine.
Preferably, the drug is a drug that inhibits pathological pain.
Still more preferably, the drug is a drug for inhibiting pathological pain, which takes the Kif11 gene as an intervention target.
Preferably, the drug is a drug that inhibits pain by transfecting nerve cells.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a recombinant adeno-associated virus rAAV9-ZsGreen-ShRNA-Kif11 containing a rat Kif11 gene interference shRNA sequence, which has the function of transfecting dorsal root ganglion nerve cells cultured in vitro, has the function of transfecting the dorsal root ganglion nerve cells of living animals, and has the function of inhibiting pathological pain sensitivity reaction, so the recombinant adeno-associated virus rAAV9-ZsGreen-ShRNA-Kif11 has good application prospect in the aspect of pain treatment medicines.
Drawings
FIG. 1 is a diagram of rat dorsal root ganglion nerve cells transfected and cultured in vitro by recombinant adeno-associated virus rAAV9-ZsGreen-shRNA-Kif 11; wherein, an arrow indicates a fluorescent group ZsGreen which can indicate transfected in vitro cultured nerve cells;
FIG. 2 is a diagram of a recombinant adeno-associated virus rAAV9-ZsGreen-shRNA-Kif11 transfected living animal dorsal root ganglion nerve cells, wherein an arrow indicates a fluorophore ZsGreen which can indicate transfected in vivo nerve cells;
FIG. 3 is a graph showing the effect of rAAV9-ZsGreen-shRNA-Kif11 in inhibiting Complete Freund's Adjuvant (CFA) -induced nociception. Wherein, the first arrow is the injection time of the dorsal root ganglion of the virus, the second arrow is the injection time of the sole of the CFA, and in the figure: * P <0.05; * P <0.01; * P <0.001.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in other sequences than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
1. preparation of recombinant adeno-associated virus rAAV9-ZsGreen-shRNA-Kif11
The method comprises the following steps: introducing a rat Kif11 gene shRNA sequence and an interference sequence into a rAAV9-ZsGreen-shShRNA plasmid vector, carrying out enzyme digestion identification on the rAAV9-ZsGreen-ShRNA-Kif11 interference plasmid vector, and packaging the interference vector rAAV9-ZsGreen-ShRNA-Kif11 recombinant 9-type adeno-associated virus.
shRNA nucleotide sequence against Kif11 gene, sense strand:
5’-GATCCGTGCTGCAAGGATCGATTGATTTCAAGAGAATCAATCGATCCTTGCAGCACTTTTTTAGATCTA-3’；
antisense strand:
5’-AGCTTAGATCTAAAAAAGTGCTGCAAGGATCGATTGATTCTCTTGAAATCAATCGATCCTTGCAGCACG-3’。
the recombinant adeno-associated virus has high titer, and each milliliter contains 1 multiplied by 10 13 The virus transduction particle has an inhibition rate of 70% on the Kif11 gene, can inhibit the Kif11 gene expression, and has an anti-tumor effect.
Specifically, the preparation operation of the recombinant adeno-associated virus rAAV9-ZsGreen-ShRNA-Kif11 containing rat Kif11 gene interference shRNA can refer to the preparation method disclosed in the patent CN 105002181B.
2. In-vitro cultured in-vitro dorsal root ganglion nerve cells transfected by recombinant adeno-associated virus rAAV9-ZsGreen-shRNA-Kif11
Acute isolation of dorsal root ganglion neurons from adult rats (4 weeks postnatal) by conventional method, making cell suspension, plating, incubating at 37 deg.C and 5% CO 2 Culturing in an incubator. 6h, changing the solution, and recombining the adeno-associated virus (1X 10) by using rAAV9-ZsGreen-shRNA-Kif11 13 vg/ml) of neurons, at 37 ℃ 5% 2 Culturing in an incubator for 48h, changing the culture solution, placing at 37 deg.C, 5% 2 Culturing in an incubator, and detecting the transfection effect under a fluorescence microscope at 2 days, 4 days and 9 days after transfection respectively.
The results show that: after 2 days of transfection, no fluorescent indicator group ZsGreen positive neurons were detected; after 4 days and 9 days of transfection, the number and the intensity of the fluorescent indicator group ZsGreen positive neurons are gradually increased (see figure 1, an arrow in the figure shows the fluorescent group ZsGreen which can indicate transfected in vitro cultured nerve cells), and the recombinant adeno-associated virus rAAV9-ZsGreen-shRNA-Kif11 is suggested to have the function of transfecting the dorsal root ganglion nerve cells cultured in vitro.
3. Recombinant adeno-associated virus rAAV9-ZsGreen-shRNA-Kif11 transfection living animal dorsal root ganglion nerve cells
Virus transfection of the dorsal root ganglion nerve cells of the living animals was carried out by conventional procedures. After 6% chloral hydrate was intraperitoneally injected to anesthetize rats, the left lumbar 4 to lumbar 6 dorsal root ganglia were surgically exposed, and 1ul of virus (1X 10) was slowly injected into the lumbar 4 to lumbar 6 ganglia using a 5ul microsyringe 13 vg/ml), muscle skin is sutured layer by layer, postoperative antibiotics prevent infection, and animals are raised conventionally. The transfection effect can be identified 4 weeks after virus injection. Rats were anesthetized with 4% sodium pentobarbital, opened to the chest, and intubated through the left ventricle to the ascending aorta. The blood was washed with 200ml of physiological saline, and then fixed by perfusion with 700ml of a phosphate buffer solution (pH 7.2-7.4) containing 4% paraformaldehyde. Immediately taking out the waist 4 to the dorsal root node of the waist 6 after the perfusion is finished, placing the waist 4 to the dorsal root node of the waist 6 in the same fresh fixing solution, fixing the waist for 24 hours (4 ℃), and then transferring the waist into 30 percent sucrose PB solution until the bottom is settled (4 ℃). Frozen sections were made with a Leica1800 cryostat microtome, the thickness of the slices being 30 μm. The sections were collected and washed in Phosphate Buffered Saline (PBS) for 3X 10min, then mounted, air dried, and mounted. Image acquisition was performed under a confocal microscope (Olympus FV1000, japan).
The results show that: fluorescent reporter ZsGreen positive neurons could be detected in the radicular tissue sections, as shown in fig. 2 (the arrows indicate fluorescent reporter ZsGreen, which indicates transfected in vivo nerve cells), as shown in fig. 2: the recombinant adeno-associated virus rAAV9-ZsGreen-shRNA-Kif11 has the function of transfecting dorsal root ganglion nerve cells of living animals.
4. Effect of recombinant adeno-associated virus rAAV9-ZsGreen-shRNA-Kif11 on pain behavior
The method for injecting virus into dorsal root ganglion nerve is the same as that described above. 4 weeks after transfection, inflammatory hyperalgesia was induced by plantar injection of complete Freund's adjuvant CFA (100. Mu.l, 1. 14 mechanical stimulators (von-Frey fibers) with different stimulation intensities (1.0, 2.5, 3.5, 4, 4.5, 5, 7.5, 10, 15, 20, 30, 40, 50, 60 g) were made with nylon filaments of different thicknesses and different lengths. A20 cm by 25cm transparent plexiglass cover is placed on a 30cm high wire grid mat and the rat to be tested is placed in the cover. After the rat is adapted for 30min, a tester holds von-Frey fibers to penetrate through a metal wire grid to stimulate the central parts of the soles at two sides of the rat respectively, the stimulation intensity is changed from small to large, each intensity is repeatedly stimulated for 10 times (each time at an interval of 3-5 s), and the intensity of the foot contraction reflex which is more than 50 percent is determined as the response threshold of the rat to the mechanical stimulation.
The results are shown in FIG. 3 (in the figure, the first arrow is the injection time of the dorsal root ganglion of the virus, and the second arrow is the injection time of the plantar CFA), and indicate that: compared with the scramble control group, the recombinant adeno-associated virus rAAV9-ZsGreen-shRNA-Kif11 can obviously inhibit pain sensitivity behaviors, and the recombinant adeno-associated virus rAAV9-ZsGreen-shRNA-Kif11 is prompted to have the effect of inhibiting pathological pain sensitivity reaction and has application prospects in the aspect of pain treatment drugs.
In conclusion, the rat Kif11 gene interference shRNA provided by the invention has the function of transfecting dorsal root ganglion nerve cells cultured in vitro; has the function of transfecting the dorsal root ganglion nerve cells of the living animals; has the function of inhibiting pathological hyperalgesia and has application prospect in the aspect of pain treatment medicines.
The above contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention should not be limited thereby, and any modification made on the basis of the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.
Claims (2)
1. The application of the recombinant adeno-associated virus rAAV9-ZsGreen-ShRNA-Kif11 based on the Kif11 gene in preparing an anti-pain medicament is characterized in that the medicament is a medicament for inhibiting pathological pain caused by inflammatory pain sensitivity induced by complete Freund's adjuvant CFA, and the medicament is a medicament for inhibiting pain by transfecting dorsal root ganglion nerve cells.
2. The use of claim 1, wherein the medicament is a Kif11 gene targeted for intervention in inhibiting pathological pain caused by inflammatory hyperalgesia induced by complete freund's adjuvant CFA.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946653A (en) * | 2015-07-03 | 2015-09-30 | 第四军医大学唐都医院 | Small-interfering RNA sequence of CHRM3 gene and application of sequence |
| CN105002181A (en) * | 2015-07-20 | 2015-10-28 | 中国人民解放军第四军医大学 | Rat Kif11 gene-interfered shRNA, recombinant adeno-associated virus thereof and anti-neoplastic application thereof |
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| WO2005014849A2 (en) * | 2003-07-03 | 2005-02-17 | Euro-Celtique, S.A. | Genes associated with responses to neuropathic pain |
| WO2005090603A2 (en) * | 2004-03-23 | 2005-09-29 | Oncotherapy Science, Inc. | Method for diagnosing non-small cell lung cancer |
| US20140170157A1 (en) * | 2011-06-15 | 2014-06-19 | Glaxosmithkline Intellectual Property (No.2) Limited | Method of selecting therapeutic indications |
| US20180141939A1 (en) * | 2016-11-22 | 2018-05-24 | Gilead Sciences, Inc. | Solid forms of a bet inhibitor |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946653A (en) * | 2015-07-03 | 2015-09-30 | 第四军医大学唐都医院 | Small-interfering RNA sequence of CHRM3 gene and application of sequence |
| CN105002181A (en) * | 2015-07-20 | 2015-10-28 | 中国人民解放军第四军医大学 | Rat Kif11 gene-interfered shRNA, recombinant adeno-associated virus thereof and anti-neoplastic application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Inhibition of kinesin-5, a microtubule-based motor protein, as a strategy for enhancing regeneration of adult axons;Shen Lin et al.;《Traffic》;20110117;第12卷(第3期);第269页摘要部分；第283页第1段 * |
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