Application of long-chain non-coding RNA GAS5 in preparation of drug for promoting nerve regeneration and repairing nerve injury
A long-chain non-coding, nerve injury technology, applied in the field of nerve regeneration and repair, can solve the problem of unclear function and mechanism
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Embodiment 1
[0028] Example 1: Expression and localization of GAS5 in DRG tissue after sciatic nerve injury
[0029] 1. DRG tissue RNA extraction and qRT-PCR
[0030] Adult healthy male C57BL / 6J mice weighing 20 g were randomly divided into 5 groups with 6 mice in each group. After compound anesthesia, the skin of the left lower limb was incised to expose the sciatic nerve parallel to the midsection of the femur on the left side, which was pinched with toothless forceps for 30 seconds, and then the skin was sutured. After the operation, the animals were fed under suitable conditions to relieve their pain, with 12 hours of light every day, and free access to water and food. 0d, 3h, 9h, 1d, 4d, 7d after sciatic nerve crushing in rats, L4~L6 DRG tissues on the crushing side Reagent (Invitrogen) manual was used to extract tissue RNA. After reverse transcription, the PrimeScript RT-PCR Kit (Takara) was used for qRT-PCR, and the operation was carried out according to the kit instructions (...
Embodiment 2
[0038] Example 2: In vitro interference with LncRNAGAS5 in DRG cells to promote neuronal axon growth 1. siRNA transfection of DRG neuron cells
[0039] Extraction and culture of primary DRG neuronal cells were performed as described in Example 1. Lipofectamine TM RNAiMAX, opti-DMEM, and GAS5 siRNA (Guangzhou Ruibo Biotech Co., Ltd.) were prepared according to the ratio of the required transfection concentration, left at room temperature for 15 minutes, and a small amount of neuron culture medium was added to the culture dish to cover the bottom of the dish, and then the prepared The siRNA mixture was dropped into the neuron cells, and after a few minutes at room temperature, gently transferred to CO 2 In the incubator, the medium was changed after 24 hours, and the cells were harvested after 48 hours.
[0040] 2. RNA extraction, reverse transcription and qRT-PCR of DRG cells
[0041] The primary DRG cells were transfected with GAS5 siRNA (siRNA1-3) and its control Negative ...
Embodiment 3
[0044] Example 3: In vivo interference with LncRNA GAS5 promotes axon regeneration
[0045] 1. siRNA injection and sciatic nerve injury in mice
[0046] A chemically modified GAS5-targeting siRNA4 (2 μl) that can be used for in vivo experiments was injected into the L4-L5 DRGs of mice through the UMP3-1 microinjection system (World Precision Company) (such as image 3 Shown in B), the sciatic nerve was crushed 2 days later, and perfused 3 days later. GAS5 siRNA4 was mixed with Invivofectamine 3.0 ReagentComplexation from Invitrogen to inhibit its degradation and improve its transfection efficiency to DRGs neurons.
[0047] 2. DRG tissue RNA extraction, reverse transcription and qRT-PCR
[0048] After injecting GAS5 siRNA in mice, the sciatic nerve was injured, and the DRG tissue was collected 3 days later, and pressed The instructions of Reagent (Invitrogen) were used to extract DRG tissue RNA, perform reverse transcription, and perform qRT-PCR. The method was the same as ...
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