WO2022007316A1 - 一种腺相关病毒及其制备在治疗可卡因成瘾的药物中的用途 - Google Patents

一种腺相关病毒及其制备在治疗可卡因成瘾的药物中的用途 Download PDF

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WO2022007316A1
WO2022007316A1 PCT/CN2020/132399 CN2020132399W WO2022007316A1 WO 2022007316 A1 WO2022007316 A1 WO 2022007316A1 CN 2020132399 W CN2020132399 W CN 2020132399W WO 2022007316 A1 WO2022007316 A1 WO 2022007316A1
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cocaine
double
mice
seq
stranded oligonucleotide
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岑小波
蒋林宏
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四川大学华西医院
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Definitions

  • the invention belongs to the field of detoxification drugs.
  • Cocaine is directly extracted from coca leaves, the compound is also known as cocaine, the chemical name is benzylecgonine. In the early days, it was commonly used as a local anesthetic or vasoconstrictor in medicine, but it was later abused due to its stimulant effects on the central nervous system.
  • Acute poisoning caused by cocaine abuse is characterized by extreme agitation, anxiety, mental disorders, blurred vision, and tremors in the limbs. Addicts often show symptoms of anxiety, burnout, and hyperexcitability that resemble delusional schizophrenia.
  • Glucosylceramide synthase belongs to the family of glycosyltransferases, which are widely present in eukaryotic membranes and are basically composed of 394 amino acids. At present, the research on UGCG and its inhibitors mainly focuses on the occurrence of tumor multidrug resistance mechanism, and there is no report on its relationship with cocaine addiction.
  • the problem to be solved by the present invention is to provide a recombinant virus that can inhibit the expression of Ugcg, and to provide the use of the virus in the treatment of cocaine addiction.
  • target sequence refers to a sequence located in the genome that is targeted and recognized by siRNA formed by in vivo processing and cleavage of shRNA.
  • shRNA its sequence is the RNA form of SEQ ID NO.3 or 4.
  • a double-stranded oligonucleotide whose sequence includes the sequence described in SEQ ID NO.1.
  • the aforementioned double-stranded oligonucleotide is characterized in that: it is a double-stranded oligonucleotide formed by base complementary pairing of the single-stranded oligonucleotides whose sequences are as described in SEQ ID NO.3 and SEQ ID NO.4 acid.
  • a recombinant plasmid containing the aforementioned double-stranded oligonucleotide A recombinant plasmid containing the aforementioned double-stranded oligonucleotide.
  • aforementioned recombinant plasmid is a recombinant plasmid obtained by inserting the aforementioned double-stranded oligonucleotide into an RNA interference vector.
  • any single strand of the double-stranded oligonucleotide or its RNA form is any single strand of the double-stranded oligonucleotide or its RNA form.
  • RNA form refers to a sequence obtained by substituting U bases for T bases in a DNA sequence.
  • the recombinant virus is an adeno-associated virus.
  • shRNA or recombinant virus that reduces the expression of glucosylceramide synthase gene in the preparation of a drug for treating cocaine addiction.
  • the shRNA is the aforementioned shRNA
  • the recombinant virus is the aforementioned recombinant virus.
  • the shRNA, double-stranded oligonucleotide, recombinant plasmid and recombinant virus of the present invention can reduce the production of GCS protein and inhibit the synthesis of downstream GlcCer (glucosylceramide) by inhibiting the expression of Ugcg gene, thereby reducing the behavioral sensitization and conditions caused by cocaine sexual position preference provides a powerful tool for the treatment of cocaine addiction, with promising application prospects.
  • Figure 1 RNAi vector.
  • FIG. 5 Cocaine-induced behavioral sensitization and conditioned place preference model in mice.
  • A Schematic diagram of the operation of the cocaine-induced behavioral sensitization model.
  • B Cocaine significantly enhanced spontaneous activity behavior in mice, t-test, ***p ⁇ 0.001.
  • C Schematic diagram of the operation of the cocaine-induced conditioned place preference model.
  • FIG. 6 Increased expression of GCS in the nucleus accumbens.
  • Figure 7 Multiple cocaine injections specifically increase nucleus accumbens GCS protein expression. Compared with the normal saline group, the level of GCS protein in the nucleus accumbens was significantly increased, and the level of Ugcg protein in the prefrontal cortex, striatum and hippocampus did not show significant changes.
  • FIG. 10 Silencing the Ugcg gene attenuates cocaine-induced behavioral sensitization effects. Two-way ANOVA analysis followed by bonferroni post-tests, *p ⁇ 0.05 and **p ⁇ 0.01.
  • AAV-shControl-Saline group, n 12;
  • AAV-shControl-Cocaine group, n 12;
  • AAV-shUgcg-Saline group, n 14;
  • AAV-shUgcg-Cocaine group, n 14.
  • Figure 11 Silencing the Ugcg gene attenuates cocaine-induced conditioned place preference effects.
  • One-way ANOVA is followed by bonferroni post-tests, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • AAV-shControl-Saline group, n 12;
  • AAV-shControl-Cocaine group, n 12;
  • AAV-shUgcg-Saline group, n 14;
  • AAV-shUgcg-Cocaine group, n 14.
  • Example 1 The present invention interferes with the construction of Ugcg expression adeno-associated virus
  • siRNA targets According to the transcript of mouse Ugcg gene, 3 to 4 siRNA targets were designed, and primer synthesis was arranged.
  • the single-stranded primer was annealed into a double-stranded oligo (oligonucleotide) sequence, and ligated into a double-enzyme-cut linearized RNA interference vector (Fig. 1) to replace the original ccdB toxicity gene.
  • the transformants were screened by colony PCR, and the positive clones screened were verified by sequencing. Sequencing to verify correct clones and high-purity plasmid extraction. The main steps are as follows:
  • Design siRNA targets arrange primer synthesis, and select the targets shown in Table 1 after screening.
  • the primer sequences are shown in Table 2.
  • most regions of shUgcg-F and shUgcg-R are reverse complementary, which can form a double-strand with sticky ends;
  • shControl-F is reverse-complementary to most regions of shControl-R, which can form a double-strand with sticky ends .
  • the uppercase bold font is the Stem region, the uppercase italic font is the Loop region, and the lowercase part is the terminal adapter sequence (including the transcription termination sequence and the restriction enzyme cleavage site sequence).
  • the synthesized primers were dissolved in oligo annealing buffer to 20 ⁇ M, and 30 ⁇ l of each complementary single strand was mixed. Then, the mixture was heated in a water bath at 95° C. for 5 min, and then the water bath was opened and cooled to room temperature naturally at room temperature to form double-stranded oligo fragments. Take 1 ⁇ l for subsequent ligation reactions, and store the rest at -20°C.
  • the expression vector was digested with restriction endonuclease.
  • the restriction enzyme digestion reaction system was: plasmid 2 ⁇ g, 10x reaction Buffer 5 ⁇ l, each restriction enzyme 1 ⁇ l, deionized water to make up 50 ⁇ l, and incubate in a water bath at 37°C for more than 2h .
  • the digestion product was subjected to agarose gel electrophoresis to detect the effect of digestion, and the target vector band was cut from the gel after agarose gel electrophoresis, and the gel was recovered with TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0.
  • the specific process is as follows:
  • TAE buffer or TBE buffer Use TAE buffer or TBE buffer to make agarose gel, and then perform agarose gel electrophoresis on the target DNA.
  • the interfering fragment is ligated into the expression vector:
  • the ligation reaction system is shown in Table 4.
  • the annealed double-stranded oligo added to the positive control is a previously annealed and validated fragment that is the same length as the annealed double-stranded oligo added to the concatenated group, but the sequence has nothing to do with it.
  • the transformants grown on the plate were picked and resuspended in 10 ⁇ l of LB medium, and 1 ⁇ l was taken as a template for colony PCR identification.
  • the transformants were identified by colony PCR, the forward primer pAKD-F TCATCAACCCGCTCCAAGGAAT (SEQ ID NO.7) was located in the human H1 promoter sequence, the reverse primer pAKD-R CAAGCTCGAAATTAACCCTCAC (SEQ ID NO.8) was located downstream of the ccdB gene, positive A 316 bp fragment was obtained by cloning.
  • the reaction system and PCR cycling conditions are as follows:
  • the positive clones obtained after colony PCR identification were sent to a sequencing company for sequencing verification.
  • the sequencing results were compared and analyzed by Vector NTI software.
  • Preparation of host cells one day before transduction (day 1), trypsinize the cells and count the cell density, and seed them into 6-well plates according to the appropriate cell density (two cells are prepared for one sample), so that the cells on the day of transfection can be prepared. The degree of fusion reaches 30%-50%. Placed in 37 °C, 5% CO 2 humidified incubator overnight.
  • Virus transfection On the day of transduction (day 2), the virus was thawed, and 10-fold dilution series of samples were prepared, and the dilution ratio was from 10 5 to 10 9 . For each diluted sample, the virus was diluted to a total volume of 1 ml with complete medium.
  • Polybrene was added to the virus-containing medium (working concentration was 6 ⁇ g/ml diluted virus solution by adding 1 ⁇ l of polybrene) to facilitate virus infection of cells. Gently pipette to mix well. Remove the culture medium from the cells and add complete culture medium that already contains different amounts of virus. In addition, keep one well of cells without virus added as a blank control group, and the volume of culture medium added to each well should be 1 ml. Placed in 37 °C, 5% CO 2 humidified incubator overnight. One day after transfection (day 3), the virus-containing medium was removed and 2 mL of fresh complete medium was added. Place in a 37°C, 5% CO 2- saturated humidity incubator overnight for culture: Count the number of fluorescent clones 2 to 3 days after virus inoculation, and calculate the virus titer.
  • Tris-base (Solarbio, G8200)
  • RNA extraction kit (Axygen, AP-MN-P-50)
  • RNAStore sample preservation solution (Tiangen Biochemical Technology Co., Ltd., DP408)
  • HPLC isopropanol (Sigma, 34863)
  • Eliglustat hemitartrate (MedChemExpress, HY-14885A)
  • DMEM high glucose medium (Hyclone, 41966052)
  • Ultra-clean workbench (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory)
  • Multi-tube rack automatic balancing centrifuge (Changsha Xiangyi Centrifuge Instrument Co., Ltd.)
  • Constant temperature shaker (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.)
  • Ultra-clean workbench (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory)
  • the wild-type animals used in this study were all male SPF-grade healthy sexually mature (8-12 weeks old) C57BL/6J mice, provided by Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., weighing 20-22g, unmated. Breeding conditions: The general animal room of the National (Chengdu) New Drug Preclinical Safety Evaluation Center, with a temperature of 20-25 °C and a relative humidity of 55-65%. During the whole experiment, animals ate and drank freely, and the breeding environment was in accordance with the "Experimental Animals" Environment and Facilities" in the standard GB14925-2001. All animal experimental operations involved in this project meet the requirements of AAALAC, and the experimental animals should be normally raised for 3-5 days before the experiment to familiarize the mice and adapt to the environment.
  • the dosing regimen of multiple cocaine injections is mainly based on the literature dosing schedule. Specifically: intraperitoneal injection of 20 mg/kg cocaine once a day for 7 consecutive days. Half an hour after the last injection of cocaine, four brain regions of prefrontal cortex, striatum, nucleus accumbens and hippocampus were taken out with reference to the stereotaxic map of the mouse brain for Western blot detection.
  • the specific operation of multiple cocaine injections for detecting the time effect is as follows: intraperitoneal injection of 20 mg/kg cocaine once a day for 1 day, 2 days, 3 days, 5 days and 7 days respectively. Half an hour after the last injection of cocaine, the nucleus accumbens was removed with reference to the stereotaxic map of the mouse brain.
  • the dosing regimen of the cocaine-induced behavioral sensitization model in mice was based on the dosing regimen in the literature with minor modifications.
  • the behavior sensitization experimental box is made of plexiglass into four square boxes of the same size. Each box is surrounded by a smooth black plexiglass plate and the bottom is a smooth white plexiglass plate.
  • the behavior sensitization box device is shown in Figure 2. Mice were free to move in the experimental box for three days, 10 min per day. The behavioral sensitization experiment was divided into two stages. During the experiment, cocaine and normal saline were injected according to Table 6.
  • the dosing schedule is shown in Table 6.
  • the mice were placed in a behavioral sensitization box, and the activity distance of the mice within 15 minutes was recorded.
  • SPSS statistical software was used to analyze the experimental results, and the differences were expressed as mean ⁇ standard error. Two-tailed t-test analysis was used to compare the differences between the cocaine group and the normal saline group, and p ⁇ 0.05 indicated a statistical difference.
  • the mouse cocaine CPP addiction model was established with reference to the methodology and slightly modified, and the CPP operation box is shown in Figure 3.
  • the CPP experimental box is composed of two large black boxes and white boxes made of plastic partitions and a small gray box in the middle. Mice move freely in the experimental box and adapt to the environment for three days, 15 min each time.
  • the CPP experiment was divided into three phases, the first phase (day1): testing the natural preferences of mice.
  • Phase 2 (day2-day7): training phase, during which the channels between the boxes were closed with plastic partitions, cocaine was injected on days 2, 4, and 6, and mice were immediately placed in the non-preferred box, days 3, 5 , 7 days of normal saline injection, the mice were immediately put into the preference box, each time the mice stayed in the box for 15min; the third stage (day8): the test stage, at this stage, remove the plastic partition, let The mice moved freely in the box, and the time spent in the black box and the white box was recorded for 15 minutes.
  • the dosing schedule is shown in Table 7. Mice were quickly dissected within 30 minutes after the test, and the nucleus accumbens was taken out for subsequent testing.
  • results were expressed by the time difference statistics during the conditioned place preference test, the dwell time in the preference box minus the dwell time difference in the non-preference box as the post-induction preference value, and the results were expressed by comparing the time difference with the natural preference state.
  • SPSS statistical software was used to analyze the experimental results, and the differences were expressed as mean ⁇ standard error.
  • Two-tailed t-test analysis was used to compare the differences between the cocaine group and the normal saline group, and p ⁇ 0.05 indicated a statistical difference.
  • Cervical vein cannulation retention is the basis for the establishment of an autonomous drug delivery model, and is also a key step in determining the success of this model.
  • This experimental technique mainly refers to literature reports, and makes appropriate adjustments according to the laboratory conditions and experimental purposes.
  • the experimental animals were adapted to the experimental environment for 3-5 days, and they were in daily contact with the experimental personnel to avoid stress reactions in the experimental animals.
  • the jugular vein cannula is prepared with imported silicone rigid tube (outer diameter 0.48mm, inner diameter 0.40mm, length about 5mm) and matching silicone hose (length about 3mm). The hose is placed at the front of the rigid tube to avoid irritation to mice .
  • mice were anesthetized by intraperitoneal injection of chloral hydrate (10%, 10ml/kg) according to the weight of the mice, and the mouse hair was removed from the clavicle on the left side of the neck.
  • a longitudinal incision of about 1cm is made, the subcutaneous tissue is bluntly separated, and the external neck vein is removed.
  • a small amount of normal saline can be dripped during the whole process to prevent the skin and blood vessels from drying out. Cut an oblique opening in the vein with ophthalmic scissors, insert the previously prepared cannula hose end into the vein, and secure the cannula with surgical thread knots.
  • the inserted prefabricated catheter pre-fills the entire circuit with sodium heparin to prevent coagulation.
  • mice were kept warm on an electric blanket until they were awake, and then the mice were placed in clean cages, and five mice were housed together. From the second day of surgery, a small amount of heparin sodium containing antibiotics was injected through the cannula every day to ensure that the catheter was unobstructed and to prevent wound infection. The postoperative recovery period for mice is usually 7 days. Mice that have completed this procedure can be used to establish a model of autonomous cocaine administration.
  • the mouse autonomous drug delivery system adopts the mouse autonomous drug delivery system manufactured by Anlai Software Technology Co., Ltd. (Ningbo, China) ( Figure 4).
  • Each operation box is placed in a soundproof cabinet with a ventilation fan.
  • the equipment in the operation box mainly includes two nose contacts, a cage lamp, an infusion connection system, a box lamp and a food trough.
  • the drug delivery system also includes a syringe pump, an experimental animal behavior recording system, and the like.
  • FR1 fixed ratio-1
  • the system automatically pumps out a preset volume of cocaine every time the mouse touches an effective nasal contactor.
  • the FR1 program was used for training.
  • the experimental training time was 120 min/day.
  • the maximum number of injections in each round of training was 100 times.
  • Effective nose touch, right nose touch hole is invalid nose touch.
  • the cage light is turned off and the signal light is on. After the invalid nose touch, there is no response.
  • the mice were dissected within 2 hours of the last administration, and the nucleus accumbens brain region was taken out for subsequent detection.
  • Criteria for successful self-administration model the formation of conditioned reflex; the frequency of self-acquiring cocaine is stable; the number of times of drug use in animals for three consecutive days is within 10% of the average.
  • mice were weighed for 1 hour a day, and the rest of the time could only drink water freely, but no feed was provided.
  • the changes in the body weight of the mice were detected.
  • the mice whose body weight was not less than 70% of the initial body weight were selected to enter the food CPP training.
  • raw preferences were tested, followed by randomization, conditioned training and testing alternately to build food CPP models.
  • mice trained with food conditioning were first fed in the feeding cage for 1 hour, and then immediately placed in the preference box for 15 minutes of training; during non-food conditioning training, the mice were first sham fed for 1 hour (put in the feeding cage, but not given feed). , and then immediately placed in the non-preferred box for 15 minutes of training.
  • the animals in the control group were not fed before training, and fed for 1 h after the training.
  • results were expressed by comparing the difference between the conditioned place preference test periods, the dwell time in the preference box minus the dwell time in the non-preference box as the post-induced preference, compared with the time difference in natural preference.
  • SPSS statistical software was used to analyze the experimental results, and the differences were expressed as mean ⁇ standard error.
  • the differences between the food-induced group and the control group were compared by two-tailed t-test analysis, and p ⁇ 0.05 indicated a statistical difference.
  • mice were quickly killed by neck dissection within the predetermined dissection time, and then the brain was quickly separated, rinsed three times with 4°C normal saline, and the nucleus accumbens was separated and removed according to the brain anatomy atlas. Put the nucleus accumbens directly into the prepared 1.5ml EP tube, then quickly freeze it in liquid nitrogen, and finally store the tissue at -80°C for subsequent testing.
  • the main steps are: first, dilute BSA to a concentration gradient of 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.025, 0 mg/ml; then The extracted protein supernatant was diluted 30 times, and 20 ⁇ l of the diluted BSA and protein samples were added to the corresponding wells of a 96-well plate respectively. Add 200 ⁇ l of working solution to each well and incubate at 37°C for 20-25min in the dark. Subsequently, the absorbance value at the wavelength of 562 nm was measured with a full-wavelength microplate reader. When the linear correlation R 2 of the BSA standard curve was >0.99, it was qualified for quantification, and then the corresponding protein concentration of each sample was calculated. Samples were diluted accordingly as needed, and 5X protein loading buffer was added to each sample for a final concentration of 1X. In a boiling water bath for 5 min, aliquot and store at -20°C for later use.
  • Brain areas of synaptic plasticity induced by multiple cocaine administration include the prefrontal cortex, the nucleus accumbens, the striatum, and the hippocampus.
  • 20 mg/kg of cocaine was intraperitoneally injected at the same time point for 7 consecutive days.
  • the mice were sacrificed by de-neck, and the prefrontal cortex, vertebral cortex and vertebral column were removed.
  • the four brain regions of septal nucleus, striatum and hippocampus were detected by Western blot to detect the expression of GCS in the above four brain regions.
  • mice in the food group were given food for 1 h before each training, while the control group did not eat. All mice were given chow for 1 h. It was found that the behavioral preference of the mice in the food group was significantly reversed compared with the control group. However, the expression of GCS protein was detected by Western Blot, and it was found that there was no significant change in the food group compared with the control group (Fig. 9).
  • the recombinant adeno-associated virus obtained in Example 1 was injected into the nucleus accumbens brain region to interfere the expression of Ugcg.
  • the results showed that in the behavioral sensitization model, compared with the shControl-saline group, the behavioral sensitization effect of the shControl-cocaine group was significantly enhanced; while the behavioral sensitization effect of the shUgcg-cocaine group was significantly weakened ( Figure 10).
  • Ugcg is the synthase of GlcCer. Inhibiting the activity of Ugcg will reduce the content of GlcCer, its downstream synthetic product.
  • the adeno-associated virus of the present invention can be used for preparation and treatment of cocaine addiction, and has a good application prospect.

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Abstract

提供了靶序列为SEQ ID NO.1所示的shRNA，该shRNA序列为SEQ ID NO.3或4的RNA形式，还提供了包括SEQ ID NO.1序列的双链寡核苷酸及由序列如SEQ ID NO.3和SEQ ID NO.4所述的单链寡核苷酸通过碱基互补配对形成的双链寡核苷酸，携带上述双链寡核苷酸及其RNA形式的重组腺相关病毒及其在制备降低可卡因成瘾性的药物中的应用。

Description

一种腺相关病毒及其制备在治疗可卡因成瘾的药物中的用途 技术领域
本发明属于戒毒药物领域。
背景技术
可卡因(Cocaine)是从古柯叶中直接提取，化合物又称古柯碱，化学名称为苯甲基芽子碱。早期在医学上通常用作局部麻醉药或血管收缩剂，后来因其对中枢神经系统的兴奋作用而导致滥用。
可卡因滥用造成急性中毒表现为极度激动、不安、精神异常、视物不清、四肢震颤，严重者可诱发心律紊乱、全身抽搐、呼吸衰竭而致死。吸食者往往表现出焦虑、倦怠和极度兴奋等症状，酷似妄想精神分裂症。
长期使用可卡因类药物，人脑在不同脑区都出现了结构和功能性的可塑性变化。可卡因吸食导致对药物的强烈渴望会给人身心健康带来极大的危害，会导致精神颓靡、心智功能缺陷、并带来一系列并发疾病感染，甚至吸食者会不顾一切寻求和获取毒品而诱发危害社会的活动。
鉴于可卡因成瘾危害甚大，找到合适的治疗靶点和药物，治疗可卡因成瘾迫在眉睫。
葡萄糖神经酰胺合成酶(glucosylceramide synthase，GCS；基因名：Ugcg，UDP-glucose ceramide glucosyltransferase)属于糖基转移酶家族，广泛存在于真核生物的膜上，基本由394个氨基酸组成。目前关于UGCG及其抑制剂的研究主要集中在肿瘤多药耐药机制的发生上，尚未见其与可卡因成瘾的关系的报道。
发明内容
本发明要解决的问题是：提供一种可抑制Ugcg表达的重组病毒，并提供该病毒在治疗可卡因成瘾药物中的用途。
本发明的技术方案如下：
一种shRNA，其靶序列如SEQ ID NO.1所示。
术语“靶序列”指：位于基因组，被shRNA在体内加工剪切形成的siRNA所靶向识别的序列。
如前述的shRNA，其序列为SEQ ID NO.3或4的RNA形式。
一种双链寡核苷酸，其序列包括SEQ ID NO.1所述序列。
如前述的双链寡核苷酸，其特征在于：它由序列如SEQ ID NO.3和SEQ ID NO.4所述的单链寡核苷酸通过碱基互补配对形成的双链寡核苷酸。
一种重组质粒，它是含有前述双链寡核苷酸。
如前述的重组质粒，它是将前述双链寡核苷酸插入到RNA干扰载体得到的重组质粒。
一种重组病毒，所述重组病毒携带前述双链寡核苷酸或其RNA形式；
或，该双链寡核苷酸或其RNA形式的任一单链。
术语“RNA形式”指将DNA序列中的T碱基替换成U碱基得到的序列。
如前述的重组病毒，所述重组病毒是腺相关病毒。
降低葡萄糖神经酰胺合成酶基因表达的shRNA或重组病毒在制备治疗可卡因成瘾的药物中的用途。
如前述的用途，所述shRNA为前述的shRNA；
或，所述重组病毒为前述的重组病毒。
本发明的shRNA、双链寡核苷酸、重组质粒、重组病毒通过抑制Ugcg基因表达，可减少GCS蛋白的产生，抑制下游GlcCer(葡萄糖神经酰胺)合成，进而减弱可卡因导致的行为敏化和条件性位置偏爱，为治疗可卡因成瘾提供有力的工具，应用前景良好。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1RNA干扰载体。
图2小鼠行为敏化实验箱。
图3条件性位置偏好实验箱。
图4自主给药实验箱。
图5可卡因诱导小鼠行为敏化和条件性位置偏爱模型建立。(A)可卡因诱导行为敏化模型操作示意图。(B)可卡因显著增强小鼠的自发活动行为，t检验，***p<0.001。可卡因组n＝14，生理盐水组n＝14。(C)可卡因诱 导条件性位置偏爱模型操作示意图。(D)可卡因显著逆转小鼠的奖赏效应，t检验，***p<0.001。可卡因组n＝15，生理盐水组n＝15。
图6伏隔核内GCS的表达增加。(A)行为敏化效应中，可卡因组小鼠伏隔核脑区GCS的表达显著上调。t检验，*p<0.05，可卡因组n＝4，生理盐水组n＝4。(B)条件性位置偏爱行为中，可卡因组小鼠伏隔核脑区GCS的表达显著上调。t检验，*p<0.05，可卡因组n＝6，生理盐水组n＝6。
图7多次可卡因注射特异性增加伏隔核GCS蛋白表达。与生理盐水组相比，伏隔核GCS蛋白水平显著升高，前额叶皮质、纹状体和海马脑区Ugcg蛋白水平未表现出显著改变。前额叶皮质(PFC)、伏隔核(NAc)、纹状体(Striatum)、海马(Hippoampus)，t检验，*p<0.05，可卡因组n＝3，生理盐水组n＝3。
图8可卡因诱导的伏隔核GCS表达增强的时间依赖效应。腹腔连续注射可卡因，与生理盐水相比，伏隔核GCS在第3天开始表达增加具有统计学差异，并趋于稳定。t检验，*p<0.05，**p<0.01，***p<0.001。可卡因组n＝4，生理盐水组n＝4。
图9食物诱导的伏隔核GCS表达未发生显著变化。(A)通过食物诱导小鼠CPP训练，与对照组相比，食物组小鼠形成明显的奖赏效应；t检验，**p<0.01。可卡因组n＝8，生理盐水组n＝8。(B)食物CPP模型中，与对照组相比，食物组小鼠伏隔核GCS表达无显著变化；t检验，n.s无显著变化，可卡因组n＝4，生理盐水组n＝4。
图10沉默Ugcg基因减弱可卡因诱导的行为敏化效应。Two-way ANOVA analysis followed by bonferroni post-tests，*p<0.05and**p<0.01。AAV-shControl-Saline组，n＝12；AAV-shControl-Cocaine组，n＝12；AAV-shUgcg-Saline组，n＝14；AAV-shUgcg-Cocaine组，n＝14。
图11沉默Ugcg基因减弱可卡因诱导的条件性位置偏爱效应。One-way ANOVA is followed by bonferroni post-tests，*p<0.05，**p<0.01，***p<0.001。AAV-shControl-Saline组，n＝12；AAV-shControl-Cocaine组，n＝12；AAV-shUgcg-Saline组，n＝14；AAV-shUgcg-Cocaine组，n＝14。
图12沉默Ugcg基因后，回补GlcCer，可逆转沉默Ugcg诱导的行为敏化(A)和条件性位置偏爱效应(B)。Two-way ANOVA analysis followed by bonferroni post-tests，*p<0.05and**p<0.01。Saline-AAV-shControl-Vehicle(SCV)组，n＝14；Cocaine-AAV-shControl-Vehicle(CCV)组，n＝14；Cocaine-AAV-shUgcg-Vehicle(CAV)组，n＝14；Cocaine-AAV-shUgcg-GlcCer(CAG)组，n＝14。
具体实施方式
实施例1：本发明干扰Ugcg表达腺相关病毒的构建
根据mouse Ugcg基因的转录本设计3到4个siRNA靶点，安排引物合 成。将单链的引物退火成双链oligo(寡核苷酸)序列，连接入双酶切线性化的RNA干扰载体(图1)，替换掉原来的ccdB毒性基因。菌落PCR筛选转化子，筛选的阳性克隆进行测序验证。测序验证正确的克隆，进行高纯度质粒抽提。主要步骤如下：
1.干扰靶点设计和引物合成：
设计siRNA靶点，安排引物合成，经筛选后选择如表1所示的靶点。
表1 siRNA靶点序列
Figure PCTCN2020132399-appb-000001
引物序列如表2所示。表中：shUgcg-F和shUgcg-R的大部分区域反向互补，可形成带粘性末端的双链；shControl-F与shControl-R的大部分区域反向互补，可形成带粘性末端的双链。大写加粗字体为Stem区，大写斜体为Loop区，小写部分为末端接头序列(包括转录终止序列和酶切位点序列)。
表2 DNA引物片段
Figure PCTCN2020132399-appb-000002
2.引物退火形成带粘性末端的双链片段：
将合成好的引物用oligo annealing buffer溶解成20μM，互补单链各取30μl混合。然后将混合物在水浴锅中95℃加热5min，然后水浴锅开盖置室温中自然冷却至室温，形成双链oligo片段。取1μl用于后续的连接反应，其余-20℃保存。
3.线性化表达载体的制备：
用限制性内切酶对表达载体进行酶切，酶切反应体系为：质粒2μg，10x反应Buffer 5μl，限制性内切酶各1μl，去离子水补足50μl，于37℃水浴锅中孵育2h以上。酶切产物进行琼脂糖凝胶电泳检测酶切效果，并把目的载体条带从琼脂糖凝胶电泳后的胶中割下来，用TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0做胶回收。具体过程如下：
3.1.使用TAE缓冲液或TBE缓冲液制作琼脂糖凝胶，然后对目的DNA进行琼脂糖凝胶电泳。
3.2.在紫外灯下切出含有目的DNA的琼脂糖凝胶，用纸巾吸尽凝胶表面的液体。此时应注意尽量切除不含目的DNA部分的凝胶，尽量减小凝胶体积，提高DNA回收率。胶块超过300mg时，请使用多个Column进行回 收，否则严重影响收率。
3.3.切碎胶块。胶块切碎后可以加快操作步骤6的胶块溶解时间，提高DNA回收率。
3.4.称量胶块重量，计算胶块体积。计算胶块体积时，以1mg＝1μl进行计算。
3.5.向胶块中加入胶块溶解液Buffer GM，Buffer GM的加量如表3所示：
表3 Buffer GM加量
Figure PCTCN2020132399-appb-000003
3.6.均匀混合后室温15-25℃溶解胶块(胶浓度较大或比较难溶时可以在37℃加热)。此时应间断振荡混合，使胶块充分溶解(约5～10分钟)。
3.7.当凝胶完全溶解后，观察溶胶液的颜色，如果溶胶液颜色由黄色变为橙色或粉色，向上述胶块溶解液中加入3M醋酸钠溶液(pH5.2)10μl，均匀混合至溶液恢复黄色。当分离小于400bp的DNA片段时，应在此溶液中再加入终浓度为20％的异丙醇。
3.8.将试剂盒中的Spin Column安置于Collection Tube上。
3.9.将上述操作步骤7的溶液转移至Spin Column中，12,000rpm离心1分钟，弃滤液。
3.10.将700μl的Buffer WB加入Spin Column中，室温12,000rpm离心30秒钟，弃滤液。
3.11.重复操作步骤10。
3.12.将Spin Column安置于Collection Tube上，室温12,000rpm离心1分钟。
3.13.将Spin Column安置于新的1.5ml的离心管上，在Spin Column膜的中央处加入30μl灭菌蒸馏水或Elution Buffer，室温静置1分钟。
3.14.室温12,000rpm离心1分钟洗脱DNA。
4.干扰片段连接入表达载体：
连接反应体系如表4所示。
表4 连接体系
Figure PCTCN2020132399-appb-000004
于16℃连接过夜。
说明：阳性对照所加的退火的双链oligo是以前退火好的验证好用的片段，和连接组所加的退火的双链oligo长度一样，但序列无关。
5.感受态细胞的转化：
DH5α感受态细胞的转化。具体操作步骤如下：
1)具有抗生素抗性的质粒0.5～2μL放入100μL感受态细胞中，冰上30min。
2)42℃热激90s,不要摇动管，立即放在冰上，冰上1～2分钟。
3)加入1mlLB液体培养基至管中，37℃，200rpm,30min～1h,活化细菌。
4)取50菌液涂平板(如果转化效率低可以多取菌液或离心弃上清取下层涂板；涂菌棒在酒精灯上烘烤之后应搁置稍凉后再涂板，以防杀死细胞)，涂板时，感受态涂两个，有抗性，无抗性，转化后菌液涂一个有抗性。(涂板后的平板先正放一会，待菌液干燥后再倒置放于37℃培养箱中培养)
6.菌落PCR鉴定阳性转化子：
挑取平板上长出的转化子重悬于10μl LB培养液中，取1μl做模板进行菌落PCR鉴定。用菌落PCR鉴定转化子，正向引物pAKD-F TCATCAACCCGCTCCAAGGAAT(SEQ ID NO.7)位于人H1启动子序列中，反向引物pAKD-R CAAGCTCGAAATTAACCCTCAC(SEQ ID NO.8)位于ccdB基因的下游，阳性克隆得到316bp的片段。反应体系和PCR循环条件如下：
Figure PCTCN2020132399-appb-000005
7.阳性克隆送测序：
经菌落PCR鉴定后得到的阳性克隆，送测序公司进行测序验证。用Vector NTI软件比对测序结果，对测序结果进行分析。
shUgcg测序结果(SEQ ID NO.9)：
Figure PCTCN2020132399-appb-000006
Figure PCTCN2020132399-appb-000007
Control测序结果(SEQ ID NO.10)：
Figure PCTCN2020132399-appb-000008
8.经测序验证正确的阳性克隆，质粒小提
使用AxyPrep质粒DNA小量试剂盒提取。操作如下：
1)取1-4ml在LB培养基中培养过夜的菌液(若使用丰富培养基，菌液体积应减半或更少)，12,000×g离心1min，弃尽上清。
2)加250μl Buffer S1悬浮细菌沉淀，悬浮需均匀，不应留有小的菌块。
3)加250μl Buffer S2，温和并充分地上下翻转4-6次混合均匀使菌体充分裂解，直至形成透亮的溶液。此步骤不宜超过5min。
4)加350μl Buffer S3，温和并充分地上下翻转混合6-8次，12,000×g离心10min。
5)吸取步骤4中的离心上清并转移到制备管(置于2ml离心管(试剂盒内提供)中)，12,000×g离心1min，弃滤液。
6)将制备管置回离心管，加500μl Buffer W1，12,000×g离心1min，弃滤液。
7)将制备管置回离心管，加700μl Buffer W2，12,000×g离心1min，弃滤液；以同样的方法再用700μl Buffer W2洗涤一次。弃滤液。
8)将制备管置回2ml离心管中，12,000×g离心1min。
9)将制备管移入新的1.5ml离心管(试剂盒内提供)中，在制备管膜中央加60-80μl Eluent或去离子水，室温静置1min。12,000×g离心1min。
9.病毒包装
取细胞状态良好，处于对数生长期的293FT细胞，细胞计数后，按照每个10cm的培养皿5×10 6个细胞数接种于培养皿中，37℃，5％C02的培养箱中培养过夜；第二天转染前去除旧的培养液，加入5mL新鲜的含10％血清DMEM培养液；制备DNA-Lipofectamine2000复合物，以一个10cm培养皿的用量为 示范准备一支无菌的5mL离心管，先加入1.5mL无血清Opt-MEMI培养液，再加入plV/helper-SL3、pLV/helper-SL4、pLV/helper-SL5、目的质粒(各41g)，轻轻颠倒混匀准备另外一支无菌的5m离心管里，加入1.5mL无血Opti-MEM培养液和40山L的Lipofectamine200。轻轻颠倒混匀，室温孵育5分钟。5分钟后，将已稀释DNA加入到含有1lipofectamine2000的无血清Opti-MEM培养液，轻轻颠倒混匀。室温孵育20分钟；将DNA-Lipofectamine2000复合物一滴一滴地添加到293FT细胞中，轻轻地前后摇晃培养皿以混匀复合物。放置37℃、5％C0饱和湿度培养箱过夜培养；转染后一天，更换10mL含10％血清DMM培养液。放置37℃、5％CO2饱和湿度培养箱中继续培养；转染后48小时收集培养上清进行浓缩；加入10m新鲜的培养液继续培养，转染后72小时再次收集浓缩；收集浓缩条件为：3000pm低速离心5min，上清用0.45μm滤器进行过滤，以彻底去除细胞碎片；每个UT离心管装20mL液，4℃50000×g高速离心90min沉淀病毒颗粒，弃去上清，用少量HSS重悬；在UT离心管装10mL预冷的20％蔗糖溶液(HBSS溶解)，将重悬溶解好的病毒液小心加到蔗糖页面上，20℃50000×g高速离心120min沉淀病毒颗粒；弃去离心上清，用HBSS重悬病毒沉淀，即得本发明重组慢病毒LV-shPRMT1，分装进入0.5m1进口AXYGEN管中，每管100u1。分装好的病毒放置-80℃保存。
10.滴度测定
宿主细胞的准备：转导前一天(第1天)，胰酶消化细胞并计数细胞密度，按照合适的细胞密度接种到6孔板中(一个样品准备两块细胞)，能使转染当天的融合度达到30％-50％。放置37℃、5％CO 2饱和湿度培养箱过夜培养。病毒转染：转导当天(第2天)，融解病毒，准备10倍稀释系列样品，稀释倍数从10 5到10 9。对于每一个稀释样品，用完全培养液稀释病毒至总体积lml。在含有病毒的培养液中加入聚凝胺(工作浓度是6μg/ml稀释病毒液中加入1μ1聚凝胺)，以促进病毒感染细胞。轻轻吹打充分混勺。去除细胞中的培养液，加入已含有不同病毒量的完全培养液。另外，保留一孔不添加病毒的细胞，作为空白对照组，每孔加入的培养液的体积应为1ml。放置37℃、5％CO 2饱和湿度培养箱过夜培养。转染后一天(第3天)，去除含有病毒的培养液，加入2mL新鲜的完全培养液。放置37℃、5％CO 2饱和湿度培养箱过夜培养：接种病毒2～3天后计数荧光克隆数目，并计算病毒滴度。
表5 病毒滴度
Figure PCTCN2020132399-appb-000009
Figure PCTCN2020132399-appb-000010
以下用实验例的方式证明本发明的有益效果。
实验例1 本发明重组腺相关病毒AAV-shUgcg治疗可卡因成瘾
1实验试剂
本部分使用的试剂如下：
盐酸可卡因(中国食品药品检定研究院)
生理盐水(四川科伦药业股份有限公司)
RIPA裂解液(上海碧云天生物技术有限公司，P0013B)
BCA试剂盒(上海碧云天生物技术有限公司，P0010)
5×SDS-PAGE电泳上样缓冲液(上海碧云天生物技术有限公司，P0015)
7.5％SDS-PAGE试剂(广州佰合生物科技有限公司，PG111)
10％SDS-PAGE试剂(广州佰合生物科技有限公司，PG112)
12.5％SDS-PAGE试剂(广州佰合生物科技有限公司，PG113)
甲醇(上海化学试剂有限公司)
蛋白质预染marker(Thermo Scientfic，26616)
Tris-base(Solarbio，G8200)
甘氨酸(Solarbio，T8060)
抗GCS抗体(上海生工生物工程股份有限公司)
抗Tubulin抗体(Cell Signaling Technology，#15115)
辣根过氧化物酶标记的二抗(Cell Signaling Technology，#8887)
BeyoECL(上海碧云天生物技术有限公司，P0018)
Tween-20(Biorad，#1706531)
RNA提取试剂盒(Axygen，AP-MN-P-50)
异丙醇(上海化学试剂有限公司)
100％乙醇(上海化学试剂有限公司)
DEPC水(上海碧云天生物技术有限公司)
RNAStore样本保存液(天根生化科技有限公司，DP408)
BestarTM qPCR RT Kit(
Figure PCTCN2020132399-appb-000011
Bioscience，DBI-2220)
Stormstar SYBY Green qPCR Mastermix(
Figure PCTCN2020132399-appb-000012
Bioscience，DBI-2243)
HPLC甲醇(Sigma，34860)
HPLC乙酸乙酯(Sigma，650528)
HPLC异丙醇(Sigma，34863)
甲酸(Sigma，695076)
LC-MS甲酸铵(Sigma，516961)
LC-MS甲酸钠(Sigma，71539)
LC-MS亮氨酸脑啡肽(Waters)
PE(17:0/17:0)(Avanti，830756)
LPC(17:0)(Avanti，855676)
Glucosylceramide(C18:1/16:0)(Avanti，860539)
Glucosylceramide((C18:1/18:0)(Avanti，860548)
Glucosylceramide((C18:1/24:1)(Avanti，860549)
Eliglustat hemitartrate(MedChemExpress，HY-14885A)
DMEM高糖培养基(Hyclone，41966052)
胎牛血清(Hyclone，10100139)
磷酸盐缓冲液(Hyclone，10010049)
二甲基亚砜(Sigma，V900090)
Nerve Growth Factor(Thermo Scientfic，13257019)
2主要仪器
电子分析天平(Sartorius)
超净工作台(上海博迅实业有限公司医疗设备厂)
冰箱(伊莱克斯)
深低温冰箱(Thermo Scientific)
制冰机(Scotsman)
平板摇床(海门市麒麟医用仪器厂)
可调式漩涡混悬仪(SCILOGEX)
超声波破碎仪(宁波新芝生物科技股份有限公司)
恒温水浴锅(国华电器有限公司)
倒置式生物显微镜TS100(Nikon)
多管架自动平衡离心机(长沙湘仪离心机仪器有限公司)
LifeECO基因扩增仪(杭州博日科技有限公司)
电泳仪(美国Bio-Rad公司)
电泳槽(美国Bio-Rad公司)
蛋白印迹凝胶成像系统(上海勤翔科学仪器有限公司)
恒温摇床(上海智城分析仪器制造有限公司)
全波长酶标仪(Thermo Electron Corporation)
CFX96TM Real-Time System(美国Bio-Rad公司)
小鼠脑立体定位仪(深圳瑞沃德生物科技有限公司)
小鼠自发活动检测箱(深圳瑞沃德生物科技有限公司)
小鼠条件性位置偏爱检测箱(宁波安来软件科技有限公司)
小鼠自主给药检测箱(宁波安来软件科技有限公司)
深低温冰箱(Thermo Scientific)
5μl平头微量注射器(深圳瑞沃德生物科技有限公司)
二氧化碳培养箱IGO150(Jouan)
超净工作台(上海博迅实业有限公司医疗设备厂)
恒温水浴锅(国华电器有限公司)
倒置式生物显微镜TS100(Nikon)
UPLC-Qtof-MS/MS(G2-S)分离检测器(Waters)
C18色谱分离柱(Waters)
超声破碎仪(宁波新芝生物科技股份有限公司)
微量移液器(Eppendorf)
3实验方法
3.1试验动物
本研究采用的野生型动物均为雄性SPF等级健康性成熟(8-12周龄)C57BL/6J小鼠，由北京维通利华实验动物技术有限公司提供，体重20-22g，未交配过。饲养条件：国家(成都)新药临床前安全性评价中心普通动物房，温度为20-25℃，相对湿度为55-65％，整个实验过程中，动物自由摄食和饮水，饲养环境符合《实验动物环境及设施》中标准GB14925-2001。本课题涉及的所有动物实验操作均符合AAALAC要求，实验前需正常饲养实验动物3-5天，以使小鼠熟悉并适应环境。
3.2多次可卡因注射
多次可卡因注射给药主要参照文献给药方案。具体为：每天腹腔注射20mg/kg可卡因一次，连续给药7天。最后一次注射给予可卡因半小时后参照小鼠脑立体定位图谱取出前额叶皮质、纹状体、伏隔核和海马四个脑区样本，用于Western blot检测。
多次可卡因注射用于检测时间效应的具体操作为：每天腹腔注射20mg/kg可卡因一次，分别连续给药1天，2天，3天，5天，7天。最后一次注射给予可卡因半小时后参照小鼠脑立体定位图谱取出伏隔核。
3.3可卡因诱导行为敏化模型建立
可卡因诱导小鼠行为敏化模型给药方案参照文献给药方案并稍作修改。行为敏化实验箱由有机玻璃做成四个相同大小的正方形箱子，每个箱子周围是光滑的黑色有机玻璃板，底部是光滑的白色有机玻璃板，行为敏化箱装置 如图2所示。小鼠在实验箱内自由活动，适应三天，每天10min。行为敏化实验分为2个阶段，在实验过程中，按照表6注射可卡因和生理盐水。第一阶段(day0)：不做任何处理条件下，使用Noldus公司的Ethovision XT记录15min内所有小鼠运动距离，作为基础值。第二阶段(day1-day7)：每天同一时间可卡因组给予可卡因，生理盐水组给予同等剂量的生理盐水，连续7天，每天1次。给药方案如表6所示。给药后立即将小鼠放入行为敏化箱内，记录15min内小鼠的活动距离。采用SPSS统计软件分析实验结果，差值以均数±标准误表示，用双尾t检验分析法比较可卡因组和生理盐水组的差异，p<0.05表示有统计学差异。
表6 行为敏化给药方案表
Figure PCTCN2020132399-appb-000013
3.4可卡因诱导小鼠条件性位置偏爱模型(Conditioned Place Preference,CPP)
小鼠可卡因CPP成瘾模型建立参考文献方法学并稍作修改，CPP操作箱如图3所示。CPP实验箱由塑料隔板做成的黑、白两个大箱和中间灰色小箱构成，黑色大箱具有黑色四壁和圆孔粗糙地面，白色大箱是白色四壁和条纹型粗糙底面。小鼠在实验箱内自由活动，适应环境三天，每次15min。CPP实验分为3个阶段，第一阶段(day1)：测试小鼠的自然偏好。第二阶段(day2-day7)：训练阶段，在此期间，用塑料隔板封闭箱间的通道，第2，4，6天注射可卡因，立即将小鼠放入非偏爱箱，第3，5，7天注射生理盐水，立即将小鼠放入偏爱箱，每次小鼠在箱内停留的时间为15min；第三阶段(day8)：测试阶段，在此阶段，移去塑料隔板，让小鼠在箱内自由活动穿梭，并记录15min小鼠分别在黑、白箱内停留时间。给药方案如表7所示。小鼠在测试完毕30min内迅速解剖，取出伏隔核以供后续检测。
结果用条件性位置偏爱测试期间时间差值统计，偏爱箱停留时间减去非偏爱箱停留时间差值作为诱导后偏好值，结果与自然偏好状态时间差相比较来表达。采用SPSS统计软件分析实验结果，差值以均数±标准误表示，用双尾t检验分析法比较可卡因组和生理盐水组的差异，p<0.05表示有统计学差异。
表7 条件性位置偏爱给药方案表
Figure PCTCN2020132399-appb-000014
Figure PCTCN2020132399-appb-000015
3.5可卡因自主给药模型建立
3.5.1小鼠颈静脉插管滞留术
颈部静脉插管滞留术是自主给药模型建立的基础，也是决定建立该模型成功的关键步骤。本实验技术主要参照文献报道，根据本实验室条件及实验目的做出适当的调整。手术前，实验动物适应3-5天实验环境，每天与实验人员接触，避免实验动物产生应激反应。颈静脉插管采用进口硅胶硬管(外径0.48mm，内径0.40mm，长度约5mm)以及配套的硅胶软管(长度约3mm)制备，软管置于硬管前端，以免对小鼠造成刺激。
手术时，根据小鼠体重腹腔注射水合氯醛(10％，10ml/kg)麻醉小鼠，剔除颈部左侧锁骨部位鼠毛，小鼠头部朝向实验人员进行仰卧式固定，在锁骨上端开一个纵向约1cm切口，钝性分离皮下组织，拨离颈部外静脉，整个过程可滴少量生理盐水，防止皮肤、血管干燥。用眼科剪在静脉上剪出斜开口，将之前制备的插管软管端插入静脉，并用手术线打结固定插管。插入的预制导管预先使整个管路充满肝素钠，防止凝血。然后盖好导管帽，缝合颈部和背部伤口。手术结束后，将小鼠在电热毯上保温至苏醒，然后将小鼠放在干净笼里，五只合笼饲养。从手术第二天开始，每天经插管注射少量含有抗生素的肝素钠，确保导管畅通，防止伤口感染。小鼠的术后恢复期通常为7天。完成此手术的小鼠可以用来建立可卡因自主给药模型。
3.5.2小鼠自主给药(Self-administration)
小鼠自主给药系统采用安莱软件科技有限公司(中国宁波)生产制造的小鼠自主给药系统(图4)。每个操作箱均放置于有通风扇的隔音柜中，操作箱内设备主要包括两个鼻触器，一个笼灯，输液连接系统，一个箱体灯以及食物槽。另外，给药系统还包括注射泵，实验动物行为记录系统等。
在小鼠可卡因自主给药训练期间，FR1(fixed ratio-1)是最简单、最基础的训练模式，即小鼠每触碰一次有效鼻触器，系统自动泵出预设体积的可卡因。本研究中采用FR1程序进行训练，实验训练时间为120min/天，每轮训练的最大注射次数为100次，每次设备不应期为20s，注射可卡因0.75mg/kg，左侧鼻触孔为有效鼻触，右侧鼻触孔为无效鼻触。每次有效鼻触后，笼灯关闭，信号灯亮，无效鼻触后，无任何反应。确定模型建立成功后，最后一次给药2h内解剖小鼠，取出伏隔核脑区用于后续检测。
自主给药模型成功的判定标准：形成条件式反射；自主获取可卡因频率稳定；动物连续三天打药次数在其平均值的10％范围以内。
3.6食物诱导的条件性位置偏爱模型
小鼠食物CPP模型建立与可卡因诱导的CPP操作类似。在建立模型之前，首先进行7天的饥饿适应期，每次适应15min。在适应期内，小鼠每天仅可进食1h，其余时间只可自由饮水，但不提供饲料。在第七天结束后，检测小鼠的体重变化情况。在第七天末选取体重不低于起始体重70％的小鼠进入食物CPP训练。在第八天开始进行检测原始偏好，然后随机分组，进行条件化训练和测试交替地建立食物CPP模型。训练期间，食物条件化训练的小鼠先在喂食笼里进食1h，然后立即放入偏爱箱，训练15min；非食物条件化训练时先进行1h假饲(放入喂食笼，但不给予饲料)，随后立即放入非偏爱箱，训练15min。对照组动物训练前不喂食，训练完成后再统一进食1h。
结果用条件性位置偏爱测试期间差值比较，偏爱箱停留时间减去非偏爱箱停留时间差值作为诱导后偏好，与自然偏好时间差值相比较来表达。采用SPSS统计软件分析实验结果，差值以均数±标准误表示，用双尾t检验分析法比较食物诱导组和对照组的差异，p<0.05表示有统计学差异。
3.7组织分离取材
各组实验检测结束后，在预定解剖时间内快速脱颈处死小鼠，然后迅速分离大脑，用4℃生理盐水冲洗3遍后，按照脑解剖图谱分离取出伏隔核。直接将伏隔核放入准备好的1.5ml EP管中，然后迅速冻存于液氮，最后待组织收集完全保存于-80℃以备后续检测。
3.8蛋白提取与蛋白免疫印迹(Western blot)
3.8.1脑组织总蛋白提取与定量
取-80℃冻存的伏隔核，加入适量100μl RIPA裂解液，裂解液体系中加入蛋白酶抑制剂Cocktail和PMSF，置于冰上裂解15min。冰浴超声10次，5秒/次，每次间隙3秒，超声目的是破碎组织细胞，充分释放溶解蛋白。接着4℃，13000g离心15min，吸取上清液。使用BCA蛋白定量试剂盒(上海碧云天生物技术有限公司)定量蛋白浓度，主要步骤为：首先，稀释BSA至0.5，0.4，0.3，0.2，0.1，0.05，0.025，0mg/ml浓度梯度；然后将提取的蛋白上清液稀释30倍，稀释好的BSA和蛋白样品分别加20μl至96孔板相应孔内。再在各孔中加入200μl工作液，37℃避光孵育20-25min。随后用全波长酶标仪测定波长562nm时的吸光度值，当BSA标准曲线线性相关性R 2>0.99为合格定量，然后计算出对应的各样本蛋白浓度。根据需要将样本进行相应稀释，再在每个样本中加入5X的蛋白上样缓冲液，使最终浓度为1X。沸水浴5min，分装，储存于-20℃备用。
3.8.2蛋白免疫印迹与曝光
按照配胶试剂盒制备10％和7.5％的聚丙烯酰胺分离胶和5％的聚丙烯酰胺上层浓缩胶。将制备好的凝胶放入电泳槽中加入电泳缓冲液后点样。根据 各蛋白在伏隔核中的丰度，调整上样体积使样品浓度适中，每个样本槽加入蛋白样本体积约为10μl。然后使用60V电压压缩蛋白样本至分离胶界限处，待条带跑过分离界限后增加电压至80V，直至目的蛋白完全分离。随后进行转膜，将载有目的蛋白的凝胶从玻璃板上转移至带有滤纸“三明治”结构的转膜夹中，并将经过甲醇活化的PVDF膜盖在胶上，转膜电压设定为100V，转膜时间则根据蛋白分子量大小设定。转膜完成后，用TBST缓冲液配制5％的脱脂牛奶封闭液，将条带浸泡在封闭液中摇床室温封闭1小时。随后用上述封闭液将蛋白一抗按需要的比例稀释，杂交带封闭条带于一抗中，4℃过夜，第二天在37℃摇床上摇1h，再用TBST洗膜三次，每次10min。根据一抗种属来源选择相应二抗，按照1:5000稀释二抗，37℃孵育1h，结束后TBST缓冲液洗膜五次，每次10min，然后TBS缓冲液洗膜一次，10min。曝光时，按照1:1的比例配制发光A液和发光B液，避光条件下将载有目的蛋白的PVDF膜置于发光混合液中反应1min，取出PVDF膜，用滤纸吸干多余发光液。将PVDF膜放入凝胶成像系统中曝光。数据处理：将曝光得到的图片用Clinx Image Analysis系统读取各条带的灰度值，用Tubulin条带灰度值作为内参进行标准化比较。
4实验结果
4.1可卡因诱导的行为敏化和条件性位置偏爱模型建立
本实验通过连续七天腹腔注射20mg/kg可卡因(图5A、B)，检测小鼠的运动距离。结果显示，从给药第一天开始，可卡因组小鼠运动距离显著高于生理盐水组，并在第三天运动距离趋于稳定，说明行为敏化模型建立成功(图5C、D)。
4.2 GCS在行为敏化和条件性位置偏爱行为中的表达
为了进一步确认GCS在可卡因诱导的成瘾行为作用，本试验使用Western Blot技术检测GCS蛋白水平的变化。结果显示，与生理盐水组相比，在行为敏化和条件性位置偏爱奖赏效应中，GCS蛋白水平显著升高，约升高1.2倍左右(图6)。
4.3多次注射可卡因增加伏隔核脑区GCS表达
多次可卡因给药诱导的神经突触可塑性脑区包括前额叶皮质(Prefrontal cortex)、伏隔核(Nuclear accumbens)、纹状体(Striatum)和海马(Hippocampus)。为了研究多次给予可卡因对各脑区GCS表达的影响，连续7天同一时间点腹腔注射20mg/kg浓度可卡因，在最后一次给药半小时后，脱颈处死小鼠，取出前额叶皮质、伏隔核、纹状体和海马四个脑区，利用Western blot检测GCS在以上四个脑区的表达。结果显示，小鼠腹腔给予可卡因后，与生理盐水组 相比，伏隔核脑区GCS表达显著上升(p<0.05)，但是前额叶皮质、纹状体以及海马脑区GCS蛋白水平并未显著变化(图7)。因此，结果表明多次可卡因注射可以特异性增加伏隔核GCS蛋白表达。
4.4可卡因诱导的伏隔核GCS表达增加随时间延长增强
为探究GCS蛋白表达随可卡因给药时间延长产生的变化，本实验设置5个时间点，分别连续给予可卡因1天、2天、3天、5天、7天，在最后一次注射可卡因半小时后，取材得到伏隔核脑区，通过Western blot对伏隔核GCS进行检测。结果显示，与生理盐水组相比，可卡因连续腹腔给药的第1、2天没有明显变化，从第三天开始，可卡因诱导的小鼠伏隔核脑区GCS表达显著增加，且连续给药5天、7天均稳定表达(图8)。以上结果表明可卡因诱导的伏隔核GCS表达增加随可卡因给药时间延长而增加，且在给药三天后表达趋于稳定，呈现出明显的时间依赖性。
4.5伏隔核中食物诱导的条件性位置偏爱模型中GCS无显著变化
为了研究GCS是否由可卡因药物诱导引起的变化，本实验使用食物诱导小鼠的条件性位置偏爱建立模型，每次训练前给食物组小鼠进食1h，对照组则不进食，待训练结束后，所有小鼠均给予饲料1h。结果发现，与对照组相比，食物组小鼠的行为偏爱发生明显的逆转。但是，通过Western Blot检测GCS蛋白的表达，结果发现与对照组相比，食物组并未发生显著改变(图9)。
4.6沉默Ugcg表达显著减弱可卡因诱导的行为敏化、条件性位置偏爱行为学效应
使用遗传学手段，在伏隔核脑区定点注射实施例1所得重组腺相关病毒干扰Ugcg表达。结果显示，在行为敏化模型中，与shControl－生理盐水组相比，shControl－可卡因组行为敏化效应显著增强；而shUgcg－可卡因组，小鼠的行为敏化效应显著减弱(图10)。
在条件性位置偏爱中，伏隔核脑区定点注射腺相关病毒干扰Ugcg表达后，可卡因诱导的奖赏效应明显减弱(图11)。
4.7回补GlcCer(葡萄糖神经酰胺)可以逆转沉默Ugcg诱导的行为学效应
Ugcg是GlcCer的合成酶，抑制Ugcg的活性，则会导致其下游合成产物GlcCer含量降低。
使用遗传学手段，在伏隔核脑区定点注射腺相关病毒干扰Ugcg表达后，每天伏隔核定点补充GlcCer。结果显示，在行为敏化模型中，回补GlcCer，小鼠自发活动运动距离明显增加(图12A)；在条件性位置偏爱模型中，明显逆转可卡因奖赏效应的减弱(图12B)。
上述结果说明，通过注射本发明的重组腺相关病毒，可减弱可卡因导致 的行为敏化和条件性位置偏爱；该减弱的效果可以通过GlcCer回补而逆转，可见本发明的腺相关病毒是通过抑制下游GlcCer的合成而起作用的。
综上，本发明的腺相关病毒能够用于制备治疗可卡因成瘾，应用前景良好。

Claims (10)

  1. 一种shRNA，其特征在于：其靶序列如SEQ ID NO.1所示。
  2. 如权利要求1所述的shRNA，其特征在于：其序列为SEQ ID NO.3或4的RNA形式。
  3. 一种双链寡核苷酸，其特征在于：其序列包括SEQ ID NO.1所述序列。
  4. 如权利要求3所述的双链寡核苷酸，其特征在于：它由序列如SEQ ID NO.3和SEQ ID NO.4所述的单链寡核苷酸通过碱基互补配对形成的双链寡核苷酸。
  5. 一种重组质粒，其特征在于：它是含有权利要求3或4所述双链寡核苷酸。
  6. 如权利要求5所述的重组质粒，其特征在于：它是将权利要求3或4所述双链寡核苷酸插入到RNA干扰载体得到的重组质粒。
  7. 一种重组病毒，其特征在于：所述重组病毒携带权利要求3或4任一所述双链寡核苷酸或其RNA形式；
    或，该双链寡核苷酸或其RNA形式的任一单链。
  8. 如权利要求7所述的重组病毒，其特征在于：所述重组病毒是腺相关病毒。
  9. 降低葡萄糖神经酰胺合成酶基因表达的shRNA或重组病毒在制备治疗可卡因成瘾的药物中的用途。
  10. 如权利要求9所述的用途，其特征在于：所述shRNA为权利要求1或2所述的shRNA；
    或，所述重组病毒为权利要求7或8所述的重组病毒。
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