WO2021114255A1 - 一种用于研究脑-肠神经环路的方法及其应用 - Google Patents

一种用于研究脑-肠神经环路的方法及其应用 Download PDF

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WO2021114255A1
WO2021114255A1 PCT/CN2019/125268 CN2019125268W WO2021114255A1 WO 2021114255 A1 WO2021114255 A1 WO 2021114255A1 CN 2019125268 W CN2019125268 W CN 2019125268W WO 2021114255 A1 WO2021114255 A1 WO 2021114255A1
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studying
nerve circuit
enteric nerve
circuit according
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湛琴琴
李志玲
詹阳
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Shenzhen Institute of Advanced Technology of CAS
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/86—Viral vectors
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    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • This application belongs to the technical field of neurology, and specifically relates to a method for studying the brain-enteric nerve circuit and its application.
  • Social communication is an important and complex cognitive behavior.
  • the regulation of social communication ability requires specific brain functional areas to achieve.
  • the synchronicity between different brain regions reflects the way the brain works in coordination under different behaviors and tasks and the participation state of neural circuits.
  • the study of synchronization between different brain areas of the brain has been a hot topic in brain science research in recent years.
  • brain connection strength and synchronization relationship can reflect the network state between different brain regions under related tasks; in patients with mental illness, the changes in synchronization between brain regions can be used as the biological characteristics of brain diseases.
  • Many mental illnesses related to abnormal social interactions have synchronization changes between brain regions.
  • Gastrointestinal dysfunction is the most common. Compared with the general population, the prevalence of gastrointestinal diseases (such as acute constipation, diarrhea, and abdominal pain) in autistic patients is significantly increased. Usually autistic children need two times the normal dose of drugs for the treatment of their gastrointestinal diseases. In a more serious case, compared with pure neurogenic autism patients, up to four times the number of patients with intestinal diseases Of autistic people need to be hospitalized.
  • a meta-analysis of the gastrointestinal dysfunction study of 84 patients with autism found that the prevalence of constipation was 22.2%, the prevalence of diarrhea was 13%, and the prevalence of other gastrointestinal symptoms was 46.8%.
  • the brain-gut axis serves as a pathway that connects the brain and the intestine, making it possible to communicate bidirectional information between the brain and the intestine. With increasing attention to the brain-gut axis, it has also attracted widespread attention as a new target for the treatment of gastrointestinal and mental diseases (such as inflammatory bowel disease, depression and post-traumatic stress disorder).
  • the brain-gut axis includes the brain, spinal cord, autonomic nervous system (sympathetic, parasympathetic, and enteric nervous system), and adrenal axis.
  • rAAVs recombinant adeno-associated viruses
  • the purpose of this application is to provide a method for studying the brain-enteric nerve circuit and its application.
  • the present application provides a method for studying the brain-enteric nerve circuit, the method comprising: injecting a transsynaptic virus carrying a gene of interest into the intestinal wall of mammals so that it can be in the central nervous system and peripheral The nervous system simultaneously expresses the target gene and uses it to study the brain-enteric nerve circuit.
  • the brain-gut axis involves a large span of neural circuits and needs to span multiple levels of synapses, although viral vectors are used in the study of enteric neural circuits, they cannot meet the research needs of long-range neural projections.
  • the method involved in this application creatively uses the transsynaptic virus as the carrier of the target gene, and by injection into the intestinal wall of mammals, it can effectively transfect neurons and specifically express the target gene in the central nervous system at the same time.
  • neural projections can be tracked through identifiable target genes to conduct research on the brain-enteric nerve circuit that regulates social behavior.
  • the time required to achieve the expression of the target gene in this method is very short, which makes the experimental process relatively short, thereby expanding its scope of application.
  • the target gene includes a gene for marking neurons.
  • the gene for marking neurons includes an enhanced green fluorescent protein gene (eGFP).
  • eGFP enhanced green fluorescent protein gene
  • the transsynaptic virus includes pseudorabies virus (PRV).
  • PRV pseudorabies virus
  • the PRV virus used in the method involved in this application can not only reversely infect neurons, but also spread across synapses. At the same time, its expression time is very short compared to rAAVs. After the intestinal wall is injected with PRV, it usually only takes no more than 5 days The time can be projected to the cerebral cortex. Therefore, in addition to realizing the neural projection tracking of the brain-gut axis, the experiment period is greatly shortened, thereby improving the efficiency of the experiment.
  • the pseudorabies virus is PRV-152.
  • the mammals include mice, rats, guinea pigs, pigs, dogs or rabbits.
  • the method for injecting a transsynaptic virus carrying a gene of interest into the intestinal wall of a mammal includes the following steps: anesthetize the mammal and perform an abdominal laparotomy to expose the intestinal tissue; and inject the virus solution into the intestinal wall tissue ; Return the intestines and suture them.
  • the method of anesthesia is as follows: anesthetize the animal by injecting sodium pentobarbital into the abdominal cavity of the animal, fix it on the dissection table, shave the hair of the operation site and disinfect with iodophor.
  • the skin on the abdomen needs to be tightened, but not completely tight.
  • the mammal is a mouse
  • the laparotomy refers to a distance of 0.8-1.2cm (for example, 0.8cm, 0.9cm, 1.0cm, 1.1cm or 1.2cm, etc.) from the longitudinal line of the genitals on the left side of the mouse's abdomen. ) Is the starting point.
  • Make a vertical incision of 3-4cm for example, 3cm, 3.2cm, 3.5cm, 3.8cm or 4cm, etc.
  • the injection location is specifically chosen to be the place on the left side of the abdomen of the mouse about one finger away from the longitudinal line of the genitals as the starting point for laparotomy.
  • a vertical incision (about 3-4 cm) is made from the pelvis to the sternum to expose the cecum, following the cecum to the tail.
  • the extension is the colon.
  • the colon of adult mice is about 7-8cm long. Due to structural differences, the middle part of the posterior colon is usually selected as the site of virus injection, and the ink is used as a horizontal mark. The reason is that the muscular layer of the posterior segment of the colon is thicker than the anterior segment, which facilitates injection.
  • the injection is usually in the middle of the two stool masses; if the colon is full of stool, a part of the stool mass can be manually separated gently to expose the colon segment for injection.
  • the mammal is a mouse
  • the injection site is the middle part of the posterior colon of the mouse.
  • the injection volume of the virus solution is 6-10 ⁇ L, such as 6 ⁇ L, 6.5 ⁇ L, 7 ⁇ L, 7.5 ⁇ L, 8 ⁇ L, 8.5 ⁇ L, 9 ⁇ L, 9.5 ⁇ L, or 10 ⁇ L.
  • the concentration of the virus solution is 10 5-10 7 pfu/mL, such as 10 5 pfu/mL, 2x10 5 pfu/mL, 5x10 5 pfu/mL, 10 6 pfu/mL, 5x10 6 pfu/mL or 10 7 pfu/mL and so on.
  • the injection rate is 0.5-1.5 ⁇ L/min, such as 0.5 ⁇ L/min, 0.7 ⁇ L/min, 0.8 ⁇ L/min, 1.0 ⁇ L/min, 1.2 ⁇ L/min, 1.4 ⁇ L/min, or 1.5 ⁇ L/min. min etc.
  • the specific injection operation it is specifically: wipe the microinjection needle with alcohol, then suck the virus solution, gently control the colon injection site with the left index finger and thumb to keep it level; The parallel direction of the muscle is slowly injected at a slight angle, and the injection is held for about 1 minute to prevent backflow.
  • the suture includes muscle layer suture and epidermal layer suture.
  • the suture of the muscle layer adopts an intermittent suture method.
  • the suture of the epidermal layer adopts a continuous suture method.
  • the suture operation is divided into two layers, in which the muscle layer adopts the intermittent suture method and the epidermal layer adopts the continuous suture method. Only on the basis of layering can the protection of internal organs and the accuracy of suture be ensured.
  • the expression time is 110-120h (for example, 110h, 115h, 120h, etc.) after the transsynaptic virus carrying the gene of interest is injected into the intestinal wall of the mammal. Because the shorter time the target gene cannot infect the entire neural circuit, and the longer time will cause related lesions and affect the survival of mice, so the best expression effect can be achieved within the above time range.
  • this application collects the intestine and brain tissues separately, fixes them with paraformaldehyde, strips off the intestinal tissues and/or slices the brain tissues, and then performs a fluorescence microscope Observe the expression intensity of the target gene to detect the transfection of the virus to the tissue.
  • the brain tissue was taken out by cardiac perfusion, and the tissue was completely soaked in 4% PFA solution overnight. After 24h, it was soaked in 30% sucrose solution until completely dehydrated (usually It takes at least 3 days), then you can use a cryostat to slice, and then observe the expression of the target gene under a fluorescence microscope after coverslipping.
  • this application provides an application of the method for studying the brain-enteric nerve circuit as described above in studying the brain-enteric nerve circuit.
  • the present application provides an application of the method for studying the brain-enteric nerve circuit as described above in the study of social behavior.
  • the method involved in this application selects the transsynaptic virus as the viral vector, and specifically uses the intestinal wall injection method to study the formation of the brain-enteric nerve circuit that regulates social behavior, and successfully transfects enteric neurons for the first time. It can quickly reversely transfect neurons (including enteric neurons) across synapses to achieve long-range neuron projection tracking.
  • Figure 1 shows the expression of eGFP in the myenteric nerve plexus after injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8 ⁇ l, 1x10 6 pfu/ml) into the posterior segment of the colon of C57BL mice for 120 hours; the arrow indicates the positive expression of eGFP Neuron; scale bar: 50 ⁇ m.
  • PRV152-eGFP pseudorabies virus carrying green fluorescent protein
  • FIG. 2 shows that after 120 hours of injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8 ⁇ l, 1x10 6 pfu/ml) into the posterior segment of the colon of C57BL mice, eGFP was found in the paraventricular nucleus (PVN) and central amygdala (CeA) of the hypothalamus. ); the box is a partial enlarged view of PVN and CeA; scale bar: left 100 ⁇ m and right 50 ⁇ m.
  • PRV152-eGFP pseudorabies virus carrying green fluorescent protein
  • Figure 3 shows the C57BL mice injected with colon segment carrying the green fluorescent protein of pseudorabies virus (PRV152-eGFP, 8 ⁇ l, 1x10 7 pfu / ml) after 120h, eGFP expression in the myenteric plexus; scale: 100 ⁇ m.
  • PRV152-eGFP pseudorabies virus
  • Figure 4 shows the expression of eGFP in the paraventricular nucleus (PVN) of the hypothalamus after injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8 ⁇ l, 1x10 7 pfu/ml) into the posterior segment of the colon of C57BL mice for 120 hours; the box is Partial enlarged view of PVN; scale bar: 100 ⁇ m.
  • PVN paraventricular nucleus
  • PRV152-eGFP pseudorabies virus carrying green fluorescent protein
  • Figure 5 shows the expression of eGFP in the myenteric nerve plexus after 96 hours of injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8 ⁇ l, 1x10 6 pfu/ml) into the posterior segment of the colon of C57BL mice; the arrow indicates the positive expression of eGFP Neuron; scale bar: 100 ⁇ m.
  • PRV152-eGFP pseudorabies virus carrying green fluorescent protein
  • Figure 6 shows the expression of eGFP in the paraventricular nucleus (PVN) of the hypothalamus after 96 hours after injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8 ⁇ l, 1x10 6 pfu/ml) into the posterior segment of the colon of C57BL mice; the box is Partial enlarged view of PVN; scale bar: 100 ⁇ m.
  • PVN paraventricular nucleus
  • PRV152-eGFP pseudorabies virus carrying green fluorescent protein
  • This embodiment provides a method for studying the brain-enteric nerve circuit, and the method includes the following steps:
  • This embodiment provides a method for studying the brain-enteric nerve circuit, and the method includes the following steps:
  • the intestines and brain tissues of the mice in Examples 1 and 2 were collected at the same time, and the cover slips were processed to observe the expression of eGFP under a fluorescence microscope.
  • the specific operation method is: open the mouse Abdominal cavity, exposing the intestines, quickly cut off the colon and small intestine, soak in the Krebs solution containing oxygen, and process the intestines in time. Place the intestine in a silicone rubber dish filled with 1 ⁇ PBS solution, and continue to infuse oxygen. After opening the intestinal wall along the middle seam under the microscope, use a steel needle to fix it smoothly along its edge.
  • the mouse was quickly perfused with the heart, the blood in the mouse was washed with 1 ⁇ PBS solution, and the brain tissue was fixed with 4% PFA solution. After the perfusion is completed, carefully remove the intact brain tissue and preserve the brainstem, and soak in 4% PFA solution for 24 hours. Then, it was completely dehydrated with 30% sucrose solution and then embedded, and the whole brain was frozen sectioned on the embedded brain. After patching the sliced brain slices in order, add an appropriate amount of mounting tablets to cover the slices.
  • Example 1 The results of Example 1 are shown in Figures 1 and 2 ( Figure 1 shows that after 120 hours of injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8 ⁇ l, 1x10 6 pfu/ml) into the posterior segment of the colon of C57BL mice, eGFP In the myenteric plexus (MP) expression, the arrow in the figure points to the neurons expressing positive eGFP; Figure 2 shows the injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8 ⁇ l, 1x10 6 pfu/ml) 120h later, the expression of eGFP in the paraventricular nucleus (PVN) and central amygdala (CeA) of the hypothalamus).
  • PRV152-eGFP pseudorabies virus carrying green fluorescent protein
  • PVN paraventricular nucleus
  • CeA central amygdala
  • Example 2 The results of Example 2 are shown in Figures 3 and 4 (Figure 3 shows that after 120 hours of injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8 ⁇ l, 1x10 7 pfu/ml) into the posterior segment of the colon of C57BL mice, eGFP Expression in the myenteric plexus (MP); Figure 4 shows that after 120 hours of injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8 ⁇ l, 1x10 7 pfu/ml) into the posterior segment of the colon of C57BL mice, eGFP is in the hypothalamic ventricle Paranuclear (PVN) expression).
  • PVN hypothalamic ventricle Paranuclear
  • the method involved in the present application can successfully transfect enteric neurons, and it can quickly reverse transfect neurons across synapses to achieve long-range neuron projection tracking.
  • mice in Example 1 were collected 96h, 120h, and 148h after the injection was completed, and the intestines and brain tissues of the mice were collected according to the above operation in the fluorescence microscope. Observe the expression of eGFP.
  • Figure 1 shows the injection of pseudorabies virus carrying green fluorescent protein (PRV152-eGFP, 8 ⁇ l, 1x10 6 pfu/ml) into the posterior segment of the colon of C57BL mice
  • Figure 6 shows the injection of pseudorabies virus carrying green fluorescent protein (PRV152-) into the posterior part of the colon of C57BL mice.
  • eGFP 8 ⁇ l, 1x10 6 pfu/ml
  • pseudorabies virus (PRV152-eGFP) carrying green fluorescent protein (PRV152-eGFP) can be expressed in the intestinal tissue and brain tissue at the same time after 96h or 120h injected into the posterior segment of the mouse colon.
  • the post-colon injection of pseudorabies virus (PRV152-eGFP) carrying green fluorescent protein (PRV152-eGFP) 120h after eGFP expression in the hypothalamic paraventricular nucleus (PVN) is stronger than 96h, which can obtain a good effect of observing and distinguishing brain regions.

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Abstract

一种用于研究脑-肠神经环路的方法，所述方法包括：向哺乳类动物的肠壁注射携带目的基因的跨突触病毒，使其在中枢神经系统和外周神经系统同时表达目的基因，并以此研究脑-肠神经环路。所涉及的方法选用跨突触病毒作为病毒载体，并特定地对动物实施肠壁注射方式，以此来研究调控社交行为的脑-肠神经环路形成，该方法首次成功转染了肠道神经元，其可以快速逆向跨突触转染神经元，从而实现长程神经元的投射追踪。

Description

一种用于研究脑-肠神经环路的方法及其应用 技术领域
本申请属于神经学技术领域，具体涉及一种用于研究脑-肠神经环路的方法及其应用。
背景技术
社会交往是一种重要且复杂的认知行为，社会交往能力的调控需要特定的大脑功能区域来实现。而不同脑区之间的同步性反应了不同行为和任务下大脑协同工作的工作方式和神经环路的参与状态。大脑不同脑区之间的同步性研究是近几年脑科学研究热点。在正常被试人群中，脑连接强度和同步关系可以反映相关任务下不同脑区之间的网络状态；在精神疾病病人中，脑区之间同步性的变化可以作为大脑病变的生物特征。许多和社会交往异常相关的精神疾病中存在脑区之间的同步性改变。研究表明海马体与前额叶之间存在同步性，而在自闭症患者和精神分裂症患者中海马体与前额叶之间的环路连接存在功能性异常。自闭症是一种神经发育障碍性疾病，其诊断依据是存在社会互动与沟通缺失，以及具有限制性及重复性行为。这些患者给家庭带来巨大经济负担，然而这些异常行为背后的神经解剖和神经环路调控机制依然不清楚。
另外，除了中枢神经系统的一些病变，自闭症患者还常伴有其他系统并发症，由以胃肠功能障碍最为常见。与普通人群相比，自闭症患者胃肠道疾病(如急性便秘、腹泻和腹痛)的患病率显著增加。通常自闭患儿需要常人的俩倍药物剂量用于治疗其胃肠道疾病，更为严重的情况是，与单纯的神经源性自闭症患者相比，高达四倍的伴有肠道疾病的自闭人群需要接受住院治疗。一项针对84例自闭症患者胃肠道功能障碍研究的meta分析发现，便秘患病率为22.2％， 腹泻患病率为13％，其他胃肠道症状患病率为46.8％。另有研究表明，60-74％的自闭症儿童有过肠炎的患病经历。研究表明胃肠功能障碍与自闭症的社会损害程度存在相关性，且社交障碍程度与便秘程度间也存在相关性，然而导致自闭症肠道功能异常的机制亦不清楚。
在全球发病率持续增高的趋势下，探讨自闭症的发病机制已成为亟待解决的大问题。脑-肠轴作为连接大脑和肠道的通路，使脑-肠的双向信息沟通成为可能。随着对于脑-肠轴关注度的增加，其作为治疗胃肠道和精神疾病(如炎症性肠病，抑郁及创伤后应激障碍)新的靶向目标也引起了广泛关注。脑-肠轴中包括大脑、脊髓，自主神经系统(交感、副交感神经和肠道神经系统)，以及肾上腺轴等。“中枢神经系统”与第二大脑“肠道神经系统”之间存在许多信号通路以及大量相同的神经递质。脑-肠的相互作用不仅在调节胃肠功能方面发挥着重要作用，例如：营养物质的消化和吸收等；而且也影响某些中枢认知功能，例如：决策的制定或社交行为等。大量研究表明异常的脑-肠连接和/或严重影响肠道神经和中枢神经系统发育的调控因子可能是导致自闭症患者胃肠功能及行为障碍的重要因素，脑-肠神经环路的研究将对我们探究自闭症的致病成因起非常重要的作用。
病毒载体作为目前最有效的神经环路研究工具，在中枢神经系统中得到了广泛的应用，但在外周神经系统中，尤其肠神经系统，因为肠道粘膜是机体最大的天然屏障，可以抵御致病菌的侵袭，对绝大多数成功转染中枢神经系统的病毒载体存在免疫源性，从而限制了对于病毒载体的选择。目前，重组腺相关病毒(rAAVs)因其低免疫原性及低毒性，已被报道可成功转染肠神经系统，实现目的基因在肠神经系统中的高表达。但鉴于其不能跨突触感染神经元，同时其转染神经元的时间一般在三周以上，不利于实验数据的快速采集，进而大大限 制了其在脑-肠神经环路研究中的应用。其次，病毒的注射方式多种多样，常见的感染肠道组织的注射方式有口服、灌肠、腹腔注射、尾静脉注射、肠系膜动脉注射等。采用不同的病毒注射方式及注射位置也会影响到其转染的效率和表达时间。
发明内容
针对现有技术的不足，本申请的目的在于提供一种用于研究脑-肠神经环路的方法及其应用。
为达到此申请目的，本申请采用以下技术方案：
一方面，本申请提供一种用于研究脑-肠神经环路的方法，所述方法包括：向哺乳类动物的肠壁注射携带目的基因的跨突触病毒，使其在中枢神经系统和外周神经系统同时表达目的基因，并以此研究脑-肠神经环路。
因为脑-肠轴涉及的神经环路跨度大，需要跨越多级突触，所以尽管有病毒载体应用在肠道神经环路研究中，但无法满足长程神经投射的研究需求。本申请所涉及的方法创造性地以跨突触病毒为目的基因的载体，并且通过哺乳类动物肠壁注射的方式，使其可以有效转染神经元进而将目的基因特异性地同时表达在中枢神经元和外周神经元中，通过可被识别的目的基因追踪神经投射，以此进行调控社交行为的脑-肠神经环路的研究。且这种方法中实现目的基因表达所需时间很短，使得实验过程相对较短，从而扩大了它的应用范围。
优选地，所述目的基因包括标记神经元的基因。
优选地，所述标记神经元的基因包括增强型绿色荧光蛋白基因(eGFP)。
优选地，所述跨突触病毒包括伪狂犬病病毒(PRV)。
本申请所涉及的方法中所使用的PRV病毒不仅可以逆向感染神经元，而且可以跨突触传播，同时其表达时间较rAAVs来说非常短，肠壁注射PRV后，通 常仅需要不超过5天的时间就可以投射到大脑皮层。因此，在实现了脑-肠轴的神经投射追踪之外，还大大缩短了实验周期，从而提高了实验效率。
优选地，所述伪狂犬病病毒为PRV-152。
优选地，所述哺乳类动物包括小鼠、大鼠、豚鼠、猪、狗或兔。
优选地，所述向哺乳类动物的肠壁注射携带目的基因的跨突触病毒的方法包括如下步骤：将哺乳类动物麻醉后进行开腹，暴露肠道组织；将病毒溶液注射入肠壁组织；将肠道归位并缝合。
本领域技术人员公知，采用不同的病毒注射方式、不同的注射位置、注射剂量等也会影响到病毒转染的效率、表达时间和表达效果。确定合适的注射方式、注射部位、注射剂量、注射速率、病毒浓度以及表达时间是脑-肠轴研究中非常重要的一环，这也是本申请中着重去解决的问题。
所述麻醉的方式是：向动物腹腔注射戊巴比妥钠麻醉动物，将其固定于解剖台上，剃除手术部位毛发并用碘伏消毒。需要拉紧腹部皮肤，但不是完全紧绷的状态。
优选地，所述哺乳类动物为小鼠，所述开腹是指以小鼠腹部偏左侧距离生殖器纵向线0.8-1.2cm(例如0.8cm、0.9cm、1.0cm、1.1cm或1.2cm等)的地方为起点，从骨盆至胸骨做3-4cm(例如3cm、3.2cm、3.5cm、3.8cm或4cm等)的垂直切口。
注射的位置特定选择在以小鼠腹部偏左侧距离生殖器纵向线约一指的地方作为开腹起点，从骨盆至胸骨做垂直切口(约3-4厘米)暴露盲肠，顺着盲肠向尾端延伸即为结肠，成年小鼠结肠长约7-8cm，由于结构差异性，通常选择结肠后段中部作为病毒注射部位，并用墨水做水平标记。原因是结肠后段肌层较前段厚，便于注射。
另外需要注意的是，若结肠内有成形的粪便团，通常会选择两个粪便团中间的部位注射；如果结肠内充满粪便，则可轻轻手动分离一部分粪便团，露出结肠段便于注射。
优选地，所述哺乳类动物为小鼠，所述注射的部位为小鼠结肠后段中部位置。
优选地，所述病毒溶液的注射量为6-10μL，例如6μL、6.5μL、7μL、7.5μL、8μL、8.5μL、9μL、9.5μL或10μL等。
优选地，所述病毒溶液的浓度为10 5-10 7pfu/mL，例如10 5pfu/mL、2x10 5pfu/mL、5x10 5pfu/mL、10 6pfu/mL、5x10 6pfu/mL或10 7pfu/mL等。
优选地，所述注射的速率为0.5-1.5μL/min，例如0.5μL/min、0.7μL/min、0.8μL/min、1.0μL/min、1.2μL/min、1.4μL/min或1.5μL/min等。
在进行具体注射操作时，具体为：用酒精擦拭微量注射针，之后吸取病毒溶液，用左手食指与拇指轻轻控制结肠注射部位，使其保持水平；右手持针在标记远端处以与纵形肌平行的走向呈微小角度缓慢注射，停留约1min，防止回流。
此处需要注意的是：将微量注射针插入肠壁，即针可在肠壁内自由移动且清晰可见，切勿插入肠腔，然后缓慢推动注射器，注射完毕可以看到肠道表层有凸起形成，停留约1min，再将针头拔出。
优选地，所述缝合包括肌肉层缝合和表皮层缝合。
优选地，所述肌肉层缝合采用间断缝合方式。
优选地，所述表皮层缝合采用连续缝合方式。
所述缝合操作分为两层进行，其中肌肉层采用间断缝合法，表皮层采用连续缝合法，基于分层的基础上，才能保证对于内脏的保护和缝合的精度。
优选地，所述表达的时间为距离向哺乳类动物的肠壁注射携带目的基因的跨突触病毒110-120h(例如110h、115h或120h等)后。因为时间更短目的基因无法感染整个神经环路，时间更长又会引起相关病变，影响小鼠的存活，因此在上述时间范围内能取得最好的表达效果。
本申请为了探究肠道及脑组织中同时表达目的基因的效果，分别收集肠道和脑组织，经多聚甲醛固定后，分别剥离肠道组织和/或进行脑组织切片后，在荧光显微镜下观察目的基因的表达强度，从而检测病毒对组织的转染情况。其中，按照常规操作(0.01M PBS-4％PFA)进行心脏灌流取出脑组织，将组织完全浸泡于4％PFA溶液中过夜，24h后将其再浸泡于30％蔗糖溶液中至完全脱水(通常需要至少3天)，之后即可以采用冰冻切片机切片，盖片后在荧光显微镜下观察目的基因的表达情况。
另一方面，本申请提供一种如上所述的用于研究脑-肠神经环路的方法在研究脑-肠神经环路中的应用。
再一方面，本申请提供一种如上所述的用于研究脑-肠神经环路的方法在研究社交行为中的应用。
相对于现有技术，本申请具有以下有益效果：
本申请所涉及的方法选用跨突触病毒作为病毒载体，并特定地采用肠壁注射方式，以此来研究调控社交行为的脑-肠神经环路形成，首次成功转染了肠道神经元，其可以快速逆向跨突触转染神经元(包括肠道神经元)从而实现长程神经元的投射追踪。
附图说明
图1表示向C57BL小鼠结肠后段注射携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP,8μl,1x10 6pfu/ml)120h后，eGFP在肌间神经丛的表达；箭头 指示eGFP阳性表达的神经元；标尺：50μm。
图2表示向C57BL小鼠结肠后段注射携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP,8μl,1x10 6pfu/ml)120h后，eGFP在下丘脑室旁核(PVN)和中央杏仁核(CeA)的表达；方框是PVN和CeA的局部放大图；标尺：左100μm右50μm。
图3表示向C57BL小鼠结肠后段注射携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP,8μl,1x10 7pfu/ml)120h后，eGFP在肌间神经丛的表达；标尺：100μm。
图4表示向C57BL小鼠结肠后段注射携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP,8μl,1x10 7pfu/ml)120h后，eGFP在下丘脑室旁核(PVN)的表达；方框是PVN局部放大图；标尺：100μm。
图5表示向C57BL小鼠结肠后段注射携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP,8μl,1x10 6pfu/ml)96h后，eGFP在肌间神经丛的表达；箭头指示eGFP阳性表达的神经元；标尺：100μm。
图6表示向C57BL小鼠结肠后段注射携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP,8μl,1x10 6pfu/ml)96h后，eGFP在下丘脑室旁核(PVN)的表达；方框是PVN局部放大图；标尺：100μm。
具体实施方式
下面通过具体实施方式来进一步说明本申请的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本申请，不应视为对本申请的具体限制。
实施例1
本实施例提供一种用于研究脑-肠神经环路的方法，所述方法包括如下步骤：
(1)将小鼠(型号C57BL-6J)腹腔注射1％戊巴比妥钠(剂量35mg/kg)进行麻醉，将其固定于解剖台上，剃除手术部位毛发并用碘伏消毒；
(2)将小鼠腹部偏左侧距离生殖器纵向线1cm的地方作为开腹起点，从骨盆至胸骨做垂直切口4cm暴露盲肠，顺着盲肠向尾端延伸即为结肠，选择结肠后段中部位置作为病毒注射部位，并用墨水做水平标记；
(3)用酒精擦拭微量注射针，之后吸取携带eGFP基因的伪狂犬病毒溶液(PRV152-eGFP)，用左手食指与拇指轻轻控制结肠注射部位，使其保持水平；右手持针在标记远端处以与纵形肌平行的走向呈微小角度注射8μL的病毒溶液(浓度为1x10 6pfu/mL，速率为1μL/min)，注射完成后停留1min，防止回流；
(4)将肠道归位，关闭腹腔，缝合腹壁及皮肤，缝合分为俩层，肌肉层间断缝合，表皮层连续缝合；待eGFP基因进行表达。
实施例2
本实施例提供一种用于研究脑-肠神经环路的方法，所述方法包括如下步骤：
(1)将小鼠(型号C57BL-6J)腹腔注射1％戊巴比妥钠(剂量35mg/kg)进行麻醉，将其固定于解剖台上，剃除手术部位毛发并用碘伏消毒；
(2)将小鼠腹部偏左侧距离生殖器纵向线1cm的地方作为开腹起点，从骨盆至胸骨做垂直切口3cm暴露盲肠，顺着盲肠向尾端延伸即为结肠，选择结肠后段中部位置作为病毒注射部位，并用墨水做水平标记；
(3)用酒精擦拭微量注射针，之后吸取携带eGFP基因的伪狂犬病毒溶液(PRV152-eGFP)，用左手食指与拇指轻轻控制结肠注射部位，使其保持水平；右手持针在标记远端处以与纵形肌平行的走向呈微小角度注射8μL的病毒溶液(浓度为1x10 7pfu/mL，速率为0.5μL/min)，注射完成后停留1min，防止回流；
(4)将肠道归位，关闭腹腔，缝合腹壁及皮肤，缝合分为俩层，肌肉层间断缝合，表皮层连续缝合；待eGFP基因进行表达。
评价试验：
在距离注射完成后的120h时，同时收集实施例1、2中小鼠的肠道及脑组织，并进行处理盖片后在荧光显微镜下观察eGFP的表达情况，操作方法具体为：打开小鼠的腹腔，暴露肠道，快速剪下结肠和小肠，浸泡在含有氧气的Krebs溶液中，并及时处理肠道。将肠道置于盛有1×PBS溶液的硅橡胶盘中，持续通入氧气。在显微镜下沿中缝打开肠壁后，用钢针沿其边缘将其固定平整。用一次性吸管吸取PBS溶液冲净肠内容物，显微镜下用精细镊剥离小肠的黏膜和黏膜下神经层(如是结肠，需再剥离横纹肌)。剥离后结束后，用PBS溶液冲洗俩次，洗去多余组织碎片。后将其浸没于4％PFA溶液中，固定40min。固定结束后用PBS溶液反复冲洗，洗去多余的PFA溶液。用手术刀分段切开小肠，取小片组织，显微镜下在载玻片上铺展平整，滴上适量封片剂后进行盖片。
同理，取出肠道组织后迅速对小鼠进行心脏灌流，用1×PBS溶液洗净小鼠体内的血液，4％PFA溶液进行脑组织的固定。灌流完成后，小心取出完整脑组织并保留脑干，置于4％PFA溶液中浸泡24h。后用30％的蔗糖溶液使其完全脱水后进行包埋，对包埋脑进行全脑冷冻切片。将切好的脑片按顺序贴片后，滴加适量封片剂进行盖片。
在荧光显微镜下对已经处理好的肠道组织和脑片进行观察，选择可以激发EGFP的激发光，在显微镜下观察到的绿色荧光部分即为脑肠环路中被病毒感染标记上的神经元细胞。
实施例1的结果如图1及图2所示(图1表示向C57BL小鼠结肠后段注射 携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP,8μl,1x10 6pfu/ml)120h后，eGFP在肌间神经丛(MP)的表达，图中箭头所指为eGFP阳性表达的神经元；图2表示向C57BL小鼠结肠后段注射携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP,8μl,1x10 6pfu/ml)120h后，eGFP在下丘脑室旁核(PVN)和中央杏仁核(CeA)的表达)。
实施例2的结果如图3及图4所示(图3表示向C57BL小鼠结肠后段注射携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP,8μl,1x10 7pfu/ml)120h后，eGFP在肌间神经丛(MP)的表达；图4表示向C57BL小鼠结肠后段注射携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP,8μl,1x10 7pfu/ml)120h后，eGFP在下丘脑室旁核(PVN)的表达)。
由上图可知，本申请所涉及的方法能够成功转染肠道神经元，其可以快速逆向跨突触转染神经元从而实现长程神经元的投射追踪。
实施例3
为了探究取材等待时间对转染效果的影响，对实施例1中的小鼠分别在距离注射完成后的96h、120h、148h时，收集小鼠的肠道及脑组织，按照上述操作在荧光显微镜下观察eGFP的表达情况。
结果如图1、图2、图5及图6所示(其中，图5表示向C57BL小鼠结肠后段注射携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP,8μl,1x10 6pfu/ml)96h后，eGFP在肌间神经丛(MP)的表达，图中箭头所指为eGFP阳性表达的神经元；图6表示向C57BL小鼠结肠后段注射携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP,8μl,1x10 6pfu/ml)96h后，eGFP在下丘脑室旁核(PVN)的表达)。148h后小鼠不能存活，无法收集组织。
由图2和图6的对比可知：向小鼠结肠后段注射携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP)96h或120h后均能同时在肠组织和脑组织中获得表达，向小鼠结肠后段注射携带绿色荧光蛋白的伪狂犬病毒(PRV152-eGFP)120h后eGFP在下丘脑室旁核(PVN)表达效果强于96h的表达效果，可得到很好的观察和区分脑区的效果。
申请人声明，本申请通过上述实施例来说明本申请的一种用于研究脑-肠神经环路的方法及其应用，但本申请并不局限于上述实施例，即不意味着本申请必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。
以上详细描述了本申请的优选实施方式，但是，本申请并不限于上述实施方式中的具体细节，在本申请的技术构思范围内，可以对本申请的技术方案进行多种简单变型，这些简单变型均属于本申请的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本申请对各种可能的组合方式不再另行说明。

Claims (15)

  1. 一种用于研究脑-肠神经环路的方法，其包括：向哺乳类动物的肠壁注射携带目的基因的跨突触病毒，使其在中枢神经系统和外周神经系统同时表达目的基因，并以此研究脑-肠神经环路。
  2. 如权利要求1所述的用于研究脑-肠神经环路的方法，其中，所述目的基因包括标记神经元的基因。
  3. 如权利要求2所述的用于研究脑-肠神经环路的方法，其中，所述标记神经元的基因包括增强型绿色荧光蛋白基因。
  4. 如权利要求1-3中任一项所述的用于研究脑-肠神经环路的方法，其中，所述跨突触病毒包括伪狂犬病病毒。
  5. 如权利要求4所述的用于研究脑-肠神经环路的方法，其中，所述伪狂犬病病毒为PRV152。
  6. 如权利要求1-5中任一项所述的用于研究脑-肠神经环路的方法，其中，所述哺乳类动物包括小鼠、大鼠、豚鼠、猪、狗或兔。
  7. 如权利要求1-6中任一项所述的用于研究脑-肠神经环路的方法，其中，所述向哺乳类动物的肠壁注射携带目的基因的跨突触病毒的方法包括如下步骤：将哺乳类动物麻醉后进行开腹，暴露肠道组织；将病毒溶液注射入肠壁组织；将肠道归位并缝合。
  8. 如权利要求7所述的用于研究脑-肠神经环路的方法，其中，所述哺乳类动物为小鼠，所述开腹是指以小鼠腹部偏左侧距离生殖器纵向线0.8-1.2cm的地方为起点，从骨盆至胸骨做3-4cm的垂直切口。
  9. 如权利要求7或8所述的用于研究脑-肠神经环路的方法，其中，所述哺乳类动物为小鼠，所述注射的部位为小鼠结肠后段中部位置。
  10. 如权利要求9所述的用于研究脑-肠神经环路的方法，其中，所述病毒 溶液的注射量为6-10μL；
    优选地，所述病毒溶液的浓度为10 5-10 7pfu/mL；
    优选地，所述注射的速率为0.5-1.5μL/min。
  11. 如权利要求7-10中任一项所述的用于研究脑-肠神经环路的方法，其中，所述缝合包括肌肉层缝合和表皮层缝合。
  12. 如权利要求11所述的用于研究脑-肠神经环路的方法，其中，所述肌肉层缝合采用间断缝合方式；
    优选地，所述表皮层缝合采用连续缝合方式。
  13. 如权利要求1-12中任一项所述的用于研究脑-肠神经环路的方法，其中，所述表达的时间为距离向哺乳类动物的肠壁注射携带目的基因的跨突触病毒110-120h后。
  14. 如权利要求1-13中任一项所述的用于研究脑-肠神经环路的方法在研究脑-肠神经环路中的应用。
  15. 如权利要求1-13中任一项所述的用于研究脑-肠神经环路的方法在研究社交行为中的应用。
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