疾病动物模型,豚鼠实验性变态反应性神经炎模型 z-bio 2021-03-08 302

method

     1\Extraction and identification of bovine sciatic nerve myelin basic protein (MBP) "① Extraction of MBP: Modified according to London's method. Take 100g of fresh bovine sciatic nerve, separate clean under aseptic, cut into pieces, add appropriate amount of pre-cooled (-4℃) chloroform/methanol (2:1), homogenize with high-speed disperser, and add chloroform/methanol (2:1) to the homogenate ) Mix the mixture to 1000ml, stir for 12 hours at 4°C, filter and degrease with Buchner funnel. Before the precipitate is drained, take out the precipitate and add 500ml of chloroform/methanol (2:1) mixture and stir for 2 hours, the same method Suction filtration 2 times, add 500ml of pre-cooled (-4°C) acetone to the sediment, stir for 2 hours, suction filtration and repeat once. Add 1000ml of double-distilled water to the sediment, stir for 12 hours, filter with suction and repeat once, centrifuge at 4℃ (12500g/min×10min), remove the supernatant, dissolve the sediment with 180ml of double-distilled water, adjust the pH value with 1mol/L HCl To 3.00, stir for 60 minutes, and centrifuge at 4°C (12500g/min×60min). The supernatant was taken and dialyzed with distilled water, then concentrated with polyethylene glycol (PEG: molecular weight 20000), and stored in a refrigerator at -60°C for later use. The whole process mentioned above is carried out in an ice bath. ② Identification of MBP: Coomassie brilliant blue staining method was used to determine protein concentration; sodium dodecyl sulfonate (SDS)-polyacrylamide gel electrophoresis method was used to observe protein bands.

　　2 "MBP-induced EAN" Take 1ml of MBP solution (3mg/ml), complete Freund's adjuvant (CFA: BCG containing 75mg/ml 0.5ml, incomplete Freund's adjuvant 1.5ml), and place it in a mortar to homogenize and emulsify , Multi-point injection into the back of the guinea pig subcutaneously, the amount of MBP is 300μg/mouse.

　　3　EAN disease scoring standard” combined with the international EAN scoring standard, the disease degree is divided into 0 to 5 grades. Grade 0: No abnormality; Grade 1: The toes of the hind limbs drag the ground slightly; Grade 2: The toes of the hind limbs drag the ground more obviously, but no other parts are involved; Grade 3: The hind limbs are significantly dragging the ground, and the hind limbs are often biased to the same side; Grade 4: Completely paralyzed both hind limbs; Grade 5: All limbs are affected, even respiratory depression.

　　4　 Pathological Observation　 ① HE staining of nerve tissue: Guinea pigs were killed by decapitation, and the brain, cerebellum, spinal cord, spinal nerve roots and sciatic nerve were quickly taken for HE staining. ②Toluidine blue staining showed nerve myelin sheath: guinea pigs were killed by decapitation, and the sciatic nerve was taken for toluidine blue staining. ③Preparation of a single nerve fiber specimen: The nerve tissue was fixed with 10.0% formalin, and then fixed with 1.0% osmium acid. The single nerve fiber was torn under a dissecting microscope and observed under a light microscope.

　　5 Recording of motor nerve conduction velocity (MNCV) and compound muscle action potential ① Electrode placement position: the proximal stimulation electrode is inserted near the ischial nodules, the distal stimulation electrode is inserted into the 0.2cm muscle on the ankle joint, and one recording electrode is inserted In the palmar muscle, one is inserted near the metacarpophalangeal joint of the little toe, and the ground electrode is inserted under the epidermis of the thigh. ②Recording method: the guinea pig is anesthetized, first record the proximal compound electromyography (PCMAPs), adjust the position of the proximal stimulation electrode, the best electrode position when the electromyography can be induced by the stimulation of the duration of 0.05ms and the intensity of 35V, enhance the stimulation When the maximum PCMAPs are induced, increase the stimulus intensity by 50.0%, and use this value as the stimulus voltage value for 15 consecutive stimulations. The induced PCMAPs are photographed and the 15 waveforms are superimposed on a piece of film. Using the highest overlap as the standard, calculate the latency and amplitude of PCMAPs. The same method records the incubation period of the distal compound electromyography (DCMAPs) and the distal F-wave (ie the reflected wave immediately following the DCMAPs). MNCV can be calculated based on the difference between the latency of PCMAPs and DCMAPs and the distance between the two pairs of stimulation electrodes.

　　6 Spleen Lymphocyte Transformation Test" adopts a trace 3H thymidine (3H-TdR) incorporation method, and the mitogens are ConA and MBP respectively.

　　7 Detection of specific anti-MBP-IgG antibody titer in guinea pig serum" was measured by ELISA (indirect method) method.

　　8　statistical processing” The experimental data are all expressed as mean±standard deviation (), and the t test is used to compare the significance of the difference between the two groups.