松果腺對(duì)輻射免疫興奮效應(yīng)調(diào)節(jié)作用的研究

1、    松果腺對(duì)輻射免疫興奮效應(yīng)調(diào)節(jié)作用的研究        摘要目的和方法：觀察75 mGY X射線全身照射去松果腺昆明系雄性小鼠，血清皮質(zhì)酮，脾和胸腺細(xì)胞免疫功能變化及體內(nèi)、外給予褪黑素對(duì)其免疫功能的影響。結(jié)果：去松果腺小鼠經(jīng)75 mGy X射線全身照射后，血清皮質(zhì)酮水平顯著升高，脾細(xì)胞對(duì)ConA反應(yīng)性和胸腺細(xì)胞自發(fā)增殖能力明顯降低，體內(nèi)注入褪黑素或體外培養(yǎng)細(xì)胞加入褪黑素可使因去松果腺免疫功能降低而重新升高。結(jié)論：松果腺褪黑素對(duì)低劑量輻射引起的免疫興奮效應(yīng)有調(diào)節(jié)作用。主題

2、詞松果體；褪黑激素；輻射；皮質(zhì)酮；免疫，細(xì)胞 Studies of regulative function of pineal gland on radiation hormesis in theimmune system after low dose radiationLIANG Yan,ZHANG Ming,LIU Shu-ZhengInstitute of Neuroendocrinology Research,Norman Bethune University of Medical Sciences,Changchun(130021)Abstract AIM and METHOD:To

3、 observe the changes of plasma corticosterone levels,thymocyte and splenocyte cell on immune functions,the effects of pinealectomized mice injected with melatonin on immune functions and the effects of melatonin on immune functions in vitro on pinealectomized mice after whole body irradiation with 7

4、5 mGy X-rays.RESULTS：After pinealectomized mice was irradiated WBI with 75 mGy X-rays,we found levels of plasma corticosterone increased obviously,and the effects of the proliferative reaction of splenocytes to ConA and spontaneous proliferation of thymocytes was found to be decrease. But injecting

5、melatonin into pinealectomized mice or putting melatonin in the cultured cell,its immune functions might renew the enhancement.CONCLUSION：Melatonin of pineal gland might regulate the immunoenhancement induced by low dose radiation.MeSHPineal body; Melatonin; Radiation; Corticosterone; Immunity,cellu

6、lar近年來(lái)，大量文獻(xiàn)資料證明松果腺是一個(gè)重要的神經(jīng)內(nèi)分泌轉(zhuǎn)換器，可將神經(jīng)信號(hào)轉(zhuǎn)換成激素的分泌，是積極參與機(jī)體調(diào)節(jié)的機(jī)能器官1。松果腺分泌的主要激素是褪黑素(melatonin,MLT),MLT有廣泛的整合、更新等生物學(xué)作用，其中使下丘腦-垂體-腎上腺和性腺系統(tǒng)功能下調(diào)，增強(qiáng)免疫功能尤為突出，還有調(diào)節(jié)生物節(jié)律、抗衰老、抗腫瘤等作用，可能是神經(jīng)內(nèi)分泌免疫調(diào)節(jié)的重要活性物質(zhì)2，3。低劑量輻射(low dose radiation,LDR)全身照射(whole body irradiation,WBI)可引起免疫系統(tǒng)的興奮效應(yīng)，主要表現(xiàn)在以T淋巴細(xì)胞功能激活為主的細(xì)胞免疫和體液免疫功能的增強(qiáng)并可提高

7、機(jī)體的抗瘤作用4，5，在LDR誘發(fā)免疫功能增強(qiáng)的同時(shí)伴有下丘腦-垂體-腎上腺皮質(zhì)系統(tǒng)功能下調(diào)等神經(jīng)內(nèi)分泌功能的改變，神經(jīng)內(nèi)分泌對(duì)免疫系統(tǒng)的調(diào)節(jié)可能是LDR免疫興奮效應(yīng)發(fā)生的必要條件，松果腺M(fèi)LT與輻射免疫興奮效應(yīng)的關(guān)系值得研究。材料與方法一、實(shí)驗(yàn)動(dòng)物與處理：實(shí)驗(yàn)用昆明系雄性小鼠由本校實(shí)驗(yàn)動(dòng)物部提供，體重(20±2) g。應(yīng)用Philips深部X線治療機(jī)給小鼠WBI，電壓200 kV，電流10 mA，濾片銅0.5 mm，鋁1.0 mm，球靶距離212 cm，劑量率12.5 mGy/min,總劑量75 mGy。動(dòng)物處理：1.假照射組和去松果腺假照射組：將正常小鼠和去松果腺小鼠經(jīng)假照射處理

8、后做為實(shí)驗(yàn)的對(duì)照組。2.去松果腺照射組：采用激光去松果腺切除術(shù)，切除小鼠松果腺(pinealectomy,PY)，飼養(yǎng)3 d，用75 mGy X射線WBI。3.去松果腺體內(nèi)注射MLT再照射組：激光切除松果腺后，每天每只小鼠肌注MLT 1 mg/次，連續(xù)注射3 d，共3 mg，然后用75 mGy X射線WBI。二、血清皮質(zhì)酮的測(cè)定：采用本室常規(guī)方法，競(jìng)爭(zhēng)性蛋白質(zhì)結(jié)合分析法6。三、脾臟和胸腺細(xì)胞懸液制備：斷頭處死小鼠，無(wú)菌條件下取脾臟和胸腺，分別置于RPMI 1640培養(yǎng)液中，用玻片研磨法制備單細(xì)胞懸液，1 500 r/min，離心5 min，棄上清，用Hanks液洗滌細(xì)胞2次，重懸于含有10%新

9、生小牛血清的RPMI 1640培養(yǎng)液中，經(jīng)200目尼龍網(wǎng)過(guò)濾后計(jì)數(shù)，根據(jù)檢測(cè)指標(biāo)，調(diào)到所需細(xì)胞濃度。四、脾細(xì)胞對(duì)ConA反應(yīng)性測(cè)定：在96孔培養(yǎng)板上，每孔加5×106個(gè)mL脾細(xì)胞懸液100L和6 mg/mL ConA 100 L，每份樣品設(shè)自身對(duì)照，平行樣3孔，37培養(yǎng)72 h,終止培養(yǎng)前6 h，每孔加入3H-TdR 18.5 kBq/25 L，多頭細(xì)胞收集器收獲細(xì)胞，應(yīng)用1214型Reckbeta液閃儀測(cè)其counts.min-1。五、胸腺細(xì)胞自發(fā)增殖反應(yīng)測(cè)定：在96孔培養(yǎng)板上，每孔加5×106個(gè)/mL胸腺細(xì)胞懸液200L及3H-TdR 18.5 kBq/25L,平行樣3

10、孔，培養(yǎng)4 h后收獲細(xì)胞，測(cè)其counts.min-1。六、體外培養(yǎng)的脾細(xì)胞加入MLT時(shí)對(duì)ConA反應(yīng)測(cè)定：在96孔培養(yǎng)板上，每孔加入5×106個(gè)/mL脾細(xì)胞懸液100 L,20 g/mL ConA 50L,0.4 mol/L MLT 50L。37，培養(yǎng)72 h，終止前6 h,每孔加3H-TdR 18.5 kBq/25L,收獲細(xì)胞，測(cè)其counts.min-1。七、體外培養(yǎng)的胸腺細(xì)胞加MLT后自發(fā)增殖能力的測(cè)定：在96孔培養(yǎng)板上，每孔加入1×107個(gè)/mL胸腺細(xì)胞懸液100 L,0.2 mol/L MLT 100 L,平行樣3孔，每孔加入3H-TdR 18.5 kBq/25

11、L。在37，培養(yǎng)4 h，收集細(xì)胞，測(cè)其counts.min-1。結(jié)果一、低劑量輻射對(duì)去松果腺小鼠血清皮質(zhì)酮水平的影響：采用激光術(shù)切除小鼠松果腺3 d后，給予75 mGy X射線WBI，觀察停照后不同時(shí)間血清皮質(zhì)酮水平的變化。結(jié)果表明，去松果腺小鼠受照后，血清皮質(zhì)酮水平明顯高于正常假照組和去松果腺假照組。其中停照后30 min、1 h、2 h、24 h和72 h組差異非常顯著(P0.01)。去松果腺假照組血清皮質(zhì)酮含量雖然比正常假照組高，但差異不顯著，見(jiàn)表1。表1去松果腺小鼠75 mGy X射線全身照射后血清皮質(zhì)酮水平的時(shí)程變化Tab 1Changes of serum corticostero

12、ne levels of pinealectomized miceat different time after WBI with 75 mGy X-rays(±s,n=6)Time intervalsCS (g/100 mL)Sham-irradiation4.71±1.85PY-sham-irradiation6.53±3.1315 min8.69±0.3830 min9.96±0.20*1 h14.95±0.77*2 h13.01±2.63*4 h11.36±7.448 h11.20±4.65*12

13、 h6.56±3.6424 h15.06±4.19*72 h16.18±1.27*P0.05,*P0.01,vs sham-irradiation group 二、低劑量輻射對(duì)去松果腺小鼠免疫功能的影響：去松果腺小鼠，給予75 mGy X射線WBI，觀察停照后24 h和72 h免疫學(xué)參數(shù)的變化。(一)脾細(xì)胞對(duì)ConA反應(yīng)的變化：去松果腺小鼠，照射后不同時(shí)間，脾細(xì)胞對(duì)ConA的反應(yīng)能力明顯降低，見(jiàn)表2。從表中可看出，去松果腺假照組和去松果腺照射后24 h和72 h組脾細(xì)胞在ConA刺激下，3H-TdR摻入量顯著少于正常假照組(P0.05,P0.01)，去松果腺照射后

14、24 h及72 h組與去松果腺假照射組對(duì)比，差異不顯著。表2去松果腺小鼠75 mGy X射線全身照射后脾細(xì)胞對(duì)ConA反應(yīng)性變化Tab 2Changes of proliferative reaction of splenocytes to ConA inpinealectomized mice after WBI with 75 mGy X-rays (±s,n=5)Groupcounts.min-11×106 cellsSham-irradiation23 029±4 620PY-sham-irradiation16 551±3 884*24 h12

15、 625±2 160*72 h13 004±3 969*P0.05,*P0.01,vs sham-irradiation group (二)胸腺自發(fā)增殖能力的變化：去松果腺小鼠，經(jīng)75 mGy X射線WBI后，胸腺細(xì)胞3H-TdR自發(fā)摻入率如表3所示。停照后24 h、72 h和去松果腺假照射組胸腺細(xì)胞3H-TdR摻入率均低于正常假照射組，其中24 h組差異非常顯著(P0.01)，72 h組差異顯著(P0.05)，去松果腺假照組差異不顯著。表3去松果腺小鼠75 mGy X射線照射后胸腺細(xì)胞自發(fā)摻入率變化Tab 3Changes in spontaneous incorpora

16、tion of thymocytes in pinealectomizedmice after WBI with 75 mGy X-rays （±s,n=5)Groupcounts.min-11×106 cellsSham-irradiation18 468±1 507PY-sham-irradiation15 862±6 74424 h4 446±1 180*72 h12 898±5 214*P0.05,*P0.01,vs Sham-irradiation group (三)體內(nèi)注射MLT對(duì)去松果腺小鼠輻射免疫效應(yīng)的影響：去松果腺小

17、鼠，每日肌肉注射1 mg/次MLT，連續(xù)注射3 d,共3 mg，然后給予75 mGy X射線WBI，停照后24 h和72 h組脾細(xì)胞對(duì)ConA反應(yīng)能力均高于去松果腺假照組，其中72 h脾細(xì)胞在ConA刺激下3H-TdR摻入率差異顯著(P0.05)；而胸腺細(xì)胞自發(fā)摻入率明顯高于去松果腺假照組，差異非常顯著（P0.01)，與正常假照組相比差異不顯著，結(jié)果見(jiàn)表4。表4體內(nèi)注射MLT對(duì)去松果腺小鼠輻射免疫效應(yīng)的影響Tab 4Effects of pinealectomized mice injected with MLT after WBI with 75 mGy X-rays on immune f

18、unctions (counts.min-11×106 cells,±s,n=5)GroupProliferative responseof splenocytesSpontaneousincorporationof thymocytessham-irradiation26157±986021288±3532PY-sham-irradiation20896±434315675±1071*24 h28280±658824556±547672 h27589±507222760±2146*P0.05,

19、vs sham-irradiation group; P0.01,vs PY-sham irradiation group (四)MLT對(duì)體外培養(yǎng)的去松果腺照射小鼠免疫細(xì)胞功能的影響：去松果腺小鼠在接受75 mGy X射線全身照射后24 h和72 h進(jìn)行脾細(xì)胞、胸腺細(xì)胞體外培養(yǎng)時(shí)，加入MLT觀察脾細(xì)胞和胸腺細(xì)胞的功能變化，結(jié)果見(jiàn)表5，照射后24 h和72 h小鼠脾細(xì)胞3H-TdR摻入率明顯高于正常假照射組和去松果腺照射組(P0.05，P0.01)，而胸腺細(xì)胞自發(fā)增殖率明顯高于正常假照射組（P0.05)，更加顯著高于去松果腺假照射組(P0.05)。表5MLT對(duì)體外培養(yǎng)的去松果腺照射小鼠免疫細(xì)胞功

20、能的影響Tab 5Effects of MLT on immune functions in vitro culturedimmune cell on pinealectomized mice after WBI with 75 mGy X-rays (counts.min-1/1×106 cells,±s,n=5）GroupProliferative responseof splenocytesSpontaneousincorporationof thymocytesSham-irradiation26 157±9 86021 288±3 532PY-

21、sham-irradiation20 896±4 34315675±1 07124 h39175±7795*32 294±10 106*72 h37264±5234*29 017±6 627*P0.05,vs sham-irradiation group; P0.01,vs PY-sham irradiation group 討論本研究主要觀察了去松果腺小鼠，給予75 mGy X射線全身照射后，血清皮質(zhì)酮水平的變化及免疫細(xì)胞功能的變化。結(jié)果表明低劑量輻射不僅未能引起在照射正常動(dòng)物時(shí)所出現(xiàn)的腎上腺皮質(zhì)功能下調(diào)7，反而明顯增高，照射后血清皮質(zhì)酮水平一直浮動(dòng)在正常對(duì)照水平之上，1 h、24 h、72 h明顯高于正常對(duì)照組，隨時(shí)間延長(zhǎng)而呈雙峰型變化。由此推測(cè)，低劑量輻射引起的腎上腺皮質(zhì)功能變化與松果腺有密切關(guān)系，去松果腺可能解除了MLT對(duì)下丘腦-垂體-腎上腺軸的抑制作用，其雙峰型變化的機(jī)制有待進(jìn)一步研究。去松果腺小鼠75 mGy X射線全身照射后免疫功能變化