【作者】 李丽；

【导师】 司军强；

【作者基本信息】 石河子大学 ， 生理学， 2007， 硕士

【摘要】 胆囊收缩素(cholecystokinin,CCK)是一种脑肠肽,不仅存在于消化系统,在中枢和外周神经系统也有广泛分布。在中枢神经系统中,CCK主要以硫酸化的八肽胆囊收缩素(sulfated cholecystokinin octapeptide,CCK-8s)作为主要活性成份发挥其生物学效应。CCK-8s参与许多重要的生理和病理生理过程,包括行为学、焦虑、学习/记忆过程、疼痛等。CCK-8s是目前发现最强的内源性抗阿片物质,还可以降低长期应用吗啡所产生的吗啡耐受。本实验运用免疫组织化学技术和细胞内微电极记录技术,观察了福尔马林致痛后对大鼠感觉传递通路背根神经节(dorsal root ganglion,DRG)和脊髓背角感觉神经元CCK免疫反应的影响,同时观察了CCK-8s对大鼠DRG神经元膜电位的作用。所得结果如下:1、福尔马林致痛对大鼠DRG神经元CCK免疫反应阳性细胞率的影响大鼠足底注射生理盐水1 h后,注射侧和非注射侧仅有少数DRG的小神经元和大、中神经元CCK免疫反应呈弱阳性。非注射侧和注射侧DRG小神经元CCK免疫反应阳性细胞率分别为24.66%±4.81%和26.21%±4.23%,大、中神经元免疫反应阳性细胞率分别为3.99%±1.75%和4.44%±1.54%,差异均无显著性。大鼠足底注射福尔马林1 h后,注射侧DRG小神经元CCK免疫反应阳性率为60.37%±6.92%,大、中神经元免疫反应阳性率为26.96%±4.01%,较同侧生理盐水1 h组均显著增加。非注射侧DRG小神经元CCK免疫反应阳性率为25.22%±5.08%,与同侧生理盐水1 h(24.66%±4.81%)相比,差异无显著性;而大、中神经元免疫反应阳性率为11.73%±2.73%,较同侧生理盐水1 h组(3.99%±1.75%)相比,差异有显著性。注射侧与非注射侧相比,大、中神经元和小神经元CCK免疫反应阳性率差异均有显著性。在大鼠足底注射福尔马林3 h后,注射福尔马林侧DRG小神经元CCK免疫反应阳性率为49.78%±3.68%,大、中神经元免疫反应阳性率为30.17%±2.87%,较注射福尔马林1 h同侧CCK免疫反应阳性率(60.37%±6.92%和26.96%±4.01%)相比,差异均无显著性。注射侧与非注射侧相比,大、中神经元和小神经元CCK免疫反应阳性率差异均有显著性。2、福尔马林致痛对大鼠DRG神经元CCK免疫反应性的影响大鼠足底注射生理盐水1 h后,注射侧和非注射侧CCK免疫反应阳性细胞的半定量光密度均值分别是0.163±0.002和0.168±0.003,差异无显著性。大鼠足底注射福尔马林1 h后,注射侧CCK免疫反应阳性细胞的半定量光密度均值是0.236±0.012,明显高于同侧生理盐水1 h组(0.163±0.002),而非注射侧(0.173±0.002)与同侧生理盐水1 h组(0.168±0.003)相比,差异无显著性。福尔马林注射侧与同组非注射侧相比,差异有显著性。在大鼠足底注射福尔马林3 h后,注射侧DRG神经元CCK免疫反应阳性细胞的半定量光密度均值为0.209±0.006,与注射福尔马林1 h组同侧(0.236±0.012)相比差异无显著性,3 h非注射侧(0.168±0.003)与1 h的非注射侧(0.173±0.005)相比,差异也无显著性。注射侧DRG神经元CCK免疫反应阳性细胞的半定量光密度均值(0.209±0.006)较非注射侧(0.168±0.003)相比,差异有显著性。3、福尔马林致痛对大鼠脊髓神经元CCK免疫反应性的影响大鼠足底注射生理盐水后,可见极少数脊髓背角神经元呈CCK免疫反应阳性。半定量光密度均值测定,注射侧和非注射侧CCK免疫反应阳性细胞的半定量光密度均值分别是0.261±0.007和0.263±0.007,差异无显著性。大鼠足底注射福尔马林1 h后,脊髓背角神经元CCK免疫反应阳性细胞数明显增加,且注射侧阳性细胞数明显高于非注射。注射侧和非注射侧CCK免疫反应阳性细胞的半定量光密度均值分别是0.397±0.014和0.295±0.007,差异有显著性;注射侧和非注射侧明显高于同侧生理盐水1 h组(0.261±0.007和0.263±0.007),差异均有显著性。大鼠足底注射福尔马林3 h后,脊髓背角神经元CCK免疫反应阳性细胞数明显少于注射福尔马林1 h时。注射侧和非注射侧脊髓背角CCK免疫反应阳性细胞的半定量光密度均值分别是0.366±0.009和0.303±0.005,差异有显著性。注射侧和非注射侧CCK免疫反应阳性细胞的半定量光密度均值,与注射福尔马林1 h组同侧(0.397±0.014和0.295±0.007)相比有所增减,但差异均无显著性。4、CCK-8s对DRG神经元膜电位的影响灌流CCK-8s(10-8~10-6 mol/L)可使部分DRG神经元(45.6%,26/57)引起浓度依赖性的去极化。此去极化可被CCK-A受体的拮抗剂prolumide(10-4 mol/L,n=8)所组断,而CCK-B受体的拮抗剂LY225910(10-4 mol/L)对CCK-8s介导的去极化没有影响。结果提示:1)由于福尔马林致痛后大鼠DRG和脊髓背角I-II层感觉神经元CCK免疫反应阳性率和半定量光密度均值均增加,表明CCK在DRG和脊髓水平参与了痛觉信息传递的调制;2)DRG神经元上可能即存在CCK-A受体,也存在CCK-B受体。激活CCK-A受体可DRG神经元去极化反应,激活CCK-B受体则不引起DRG神经元膜电位的明显改变,表明CCK-A和CCK-B受体在DRG发挥不同的生理功能;3)DRG神经元胞浆内含有CCK神经肽,而DRG神经元胞膜上存在CCK-A和CCK-B受体,有理由推测初级感觉传入末稍突触前膜可能存在突触前CCK-A和CCK-B自身受体。更多还原

【Abstract】 CCK is a kind of brain-gut peptide that is abundantly distributed in both gastrointestinal tract and the central and peripheral nervous systems. In the central nervous system, the sulfated octapeptide fragment of cholacystokinin (CCK-8s) is engaged in numerous physiological and pathological processes including behavior, anxiety, learning/memory processes and pain. CCK-8s is one of the strongest endogenous anti-opioid substances and suppresses morphine tolerance which results from long term use of morphine. Here we observe the effect of formalin-induced pain on CCK immunoreactivity in rat sensory neurons through immunohistochemistry technique, and test the effect of CCK-8s on membrane potential of DRG neurons through intracellular recording technique. Results are as follows:1. The Effect of Formalin-induced Pain on positive rate of CCK Immunoreactivity in Rat DRG Neurons.After 1h of subcutaneous injection of saline in one paw of rats, we observe that CCK immunoreactivity in few small, moderate and big neurons on DRG are weak positive both in injection and non-injection side. Positve rate of CCK immunoreactivity of small neurons on DRG are 24.66%±4.81% and 26.21%±4.23% in injection and non-injection respectively(t=0.22,P>0.05); and positve rate of CCK immunoreactivity of moderate and big neurons on DRG are 3.99%±1.75% and 4.44%±1.54% respectively(t=0.16, P>0.05), no remarkable difference reveals.After 1h of subcutaneous injection of formalin in one paw of rats, positive rate of CCK immunoreactivity of small neurons on DRG is 60.37%±6.92% in injection side which is greater than that of saline group(t=4.21,P<0.01). and positive rate of CCK immunoreactivity of moderate and big neurons on DRG is 26.96%±4.01%, higher than that of saline group (t=5.24, P<0.01). In non-injection side, positive rate of CCK immunoreactivity of small neurons on DRG is 25.22%±5.08%, no differences reveal compared with saline group(t=0.08,P>0.05); positive rate of CCK immunoreactivity of moderate and big neurons on DRG is 11.73%±2.73% which is a remarkable difference compared with saline group(3.99%±1.75%) (t=2.38, P<0.05). Compare positive rates of CCK immunoreactivity of small neurons on DRG of injection side and non-injection side, the difference is significant (t=8.94,P<0.01), so do the moderate and big neurons on DRG(t=2.81,P<0.05).After 3h of subcutaneous injection of formalin in one paw of rats, positive rate of CCK immunoreactivity of small neurons on DRG is 49.78%±3.68% in injection side which is slightly lower than that of injection of formalin after 1h (t=1.35,P>0.05). and positive rate of CCK immunoreactivity of moderate and big neurons on DRG is 30.17%±2.87% which is slightly higher than that of injection of formalin after 1h (26.96%±4.01%) (t=0.65 P>0.05). Compare positive rates of CCK immunoreactivity of small neurons on DRG of injection side and non-injection side, the difference is apparent(t=4.36,P<0.01), so do the moderate and big nerons on DRG(t=4.44,P<0.01).2. The Effect of Formalin-induced Pain on CCK Immunoreactivity in Rat DRG Neurons.After 1h of subcutaneous injection of saline in one paw of rats, the semi-quantitative optical density average values of CCK immunoreactivity neurons on DRG are 0.163±0.002 and 0.168±0.003 in injection side and non-injection side respectively(t=1.49,P>0.05), no remarkable difference reveals.After 1h of subcutaneous injection of formalin in one paw of rats, the semi-quantitative optical density average value of CCK immunoreactivity neurons on DRG is 0.236±0.012 in injection side which is higher than that of saline group after 1h (0.163±0.002) (t=5.49,P<0.01). Compare non-injection side of formalin with non-injection side of saline, the semi-quantitative optical density average values of CCK immunoreactivity neurons on DRG, the results are similar (t=0.92,P>0.05). the value in formalin injection side is greater than that of non-injection side(t=6.65,P<0.01).After 3h of subcutaneous injection of formalin in one paw of rats, the semi-quantitative optical density average value of CCK immunoreactivity neurons on DRG is 0.209±0.006 while it is 0.236±0.012 after 1h in injection side(t=2.01,P>0.05). In non-injection side, this value of CCK immunoreactivity neurons on DRG is 0.168±0.003 while it is 0.173±0.005 after 1h (t=0.92,P>0.05). the value in injection side of formalin is (0.209±0.006) greater than that of non-injection side(0.168±0.003) (t=7.33,P<0.01).3. The Effect of Formalin-induced Pain on CCK Immunoreactivity in Spinal Cord Neurons.After subcutaneous injection of saline in one paw of rats, we observe that CCK immunoreactivity in few neurons in spinal cord is positive. The semi-quantitative optical density average values of CCK immunoreactivity neurons are 0.261±0.007 and 0.263±0.007 in injection side and non-injection side respectively(t=0.44,P>0.05).After 1h of subcutaneous injection of formalin in one paw of rats, the number of positive neurons of CCK immunoreactivity in spinal cord neurons is obviously increasing and greater than that of non-injection side. The semi-quantitative optical density average values of CCK immunoreactivity neurons are 0.397±0.014 and 0.295±0.007 in injection side and non-injection side respectively(t=8.19,P<0.01), the difference is obvious . The value(0.397±0.014) in injection side of formalin is greater than that of (0.261±0.007) injection side of saline (t=8.85,P<0.01), and the value (0.295±0.007) in non-injection side of formalin is greater than that of (0.263±0.007) non-injection side of saline (t=3.31,P<0.01).After 3h of subcutaneous injection of formalin in one paw of rats, the number of positive neurons of CCK immunoreactivity in spinal cord becomes fewer than that of after 1h. The semi-quantitative optical density average values of CCK immunoreactivity neurons are 0.366±0.009 and 0.303±0.005 in injection side and noninjection side respectively(t=8.19,P<0.01), the difference is of significance; the value(0.366±0.009) in injection side of formalin is lower than it (0.397±0.014)in injection side of formalin after 1h (t=1.86,P>0.05); and the value (0.303±0.005) in non-injection side of formalin is greater than that of(0.295±0.007)in non-injection side of formalin after 1h (t=0.96,P>0.05), but the difference is not significant.4.CCK-8s evoked membrane depolarization in rat DRG neuronsAbout 45.6% (26/57) CCK-8s (10-8~10-6 mol/L) evoked a depolarizing response. The membrane depolarization induced by CCK-8s was concentration dependent and abolished by prolumide (10-4 mol/L), a selective antagonist of CCK-A receptor (n=8). LY225910 (10-4 mol/L), a selective antagonist of CCK-B receptor (n=9), had no effect on CCK-8s evoked membrane depolarization.Results indicate:1) The positive rate and semi-quantitative optical density average value of CCK immunoreactivity neurons on DRG and in spinal dorsal horn neurons increase with formalin induced pain, this reveals that CCK participates in modulation of pain sensory conduction;2) There are possibly CCKA receptor and CCKB receptor on DRG neurons. Stimulating CCKA receptor can lead to depolarization reaction in DRG neurons, but CCKB cannot, which indicates that CCKA and CCKB play different physiological roles;3) The CCK neuropeptidein exists in the plasma of DRG neurons, and CCKA and CCKB on the membrane of DRG neurons, so we can conclude that presynaptic CCKA and CCKB receptors exist in the presynaptic membrane of primary sensory afferent endings.更多还原