梁爽 張貴育 孔杰
【摘要】 目的 探討骨髓間質干細胞的免疫原性, 評估骨髓間質干細胞活化免疫系統(tǒng)的能力。
方法 分離培養(yǎng)大鼠股骨中的骨髓間質干細胞, 擴增后分別經體外細胞刺激實驗和體內移植后評估兩個方面來評價大鼠骨髓間質干細胞的免疫原性。體外實驗采用淋巴細胞激活實驗進行, 體內實驗將骨髓間質干細胞經靜脈移植到大鼠體內, 觀察移植后大鼠的脾臟指數, 并解剖大鼠, 了解骨髓間質干細胞的致瘤性。結果 大鼠骨髓間質干細胞在體外增殖能力強, 反復傳代至10代以后, 仍能良好生長, 且生物學特征無明顯變化。各組在加入CCK-8試劑終止反應后用酶標檢測儀測得的結果顯示:觀察組為(1.515±0.737), 陽性對照組為(1.966±0.421), 陰性對照組為(1.302±0.259), 三組比較差異具有統(tǒng)計學意義(P<0.05), 觀察組與陽性對照組比較差異具有統(tǒng)計學意義(P<0.05), 陰性對照組與觀察組比較差異無統(tǒng)計學意義(P>0.05)。細胞移植后, 外源性骨髓間質干細胞進入大鼠血液系統(tǒng), 具有直接活化免疫系統(tǒng)的條件;同時隨著循環(huán)系統(tǒng)遷移, 向全身播散;在移植后不同時間點解剖大鼠, 仔細觀察各內臟器官、腦組織和骨骼肌, 組織形態(tài)基本正常, 未發(fā)現腫瘤形成。細胞移植后第8周處死大鼠, 解剖分離脾臟, 計算脾臟指數, 結果顯示, 實驗組大鼠脾臟指數(58.7±12.4)與對照組的(57.3±21.5)比較差異無統(tǒng)計學意義(P>0.05)。結論 骨髓間質干細胞在體外共培養(yǎng)體系中不能活化淋巴細胞, 經靜脈移植后在體內也不能上調宿主的免疫狀態(tài), 且無明顯致瘤性, 是用于細胞移植治療的良好種子細胞。
【關鍵詞】 骨髓間質干細胞;免疫原性;細胞移植
DOI:10.14163/j.cnki.11-5547/r.2020.02.088
Immunogenicity of bone marrow mesenchymal stem cells in rats? ?LIANG Shuang, ZHANG Gui-yu, KONG Jie. Shenzhen Baoan Hospital of Traditional Chinese Medicine, Shenzhen 518133, China
【Abstract】 Objective? ?To investigate the immunogenicity of bone marrow mesenchymal stem cells and evaluate their ability to activate the immune system. Methods? ?Bone marrow mesenchymal stem cells were isolated and cultured in vitro. After amplification, the immunogenicity of bone marrow mesenchymal stem cells was evaluated from both in vitro and in vivo. In vitro experiments were carried out using lymphocyte activation assay, and in vivo experiments were performed by transplanting bone marrow mesenchymal stem cells into rats. The spleen index of the transplanted rats was observed, and the rats were dissected to understand the tumorigenicity of bone marrow mesenchymal stem cells. Results? ?The proliferation ability of bone marrow mesenchymal stem cells in rats is strong in vitro, and they can grow well after 10 generations, and there is no obvious change in biological characteristics. After the termination of reaction with CCK-8 reagent, the results measured by enzyme-linked detector in each group showed that: the observation group was (1.515±0.737), the positive control group was (1.966±0.421), the negative control group was (1.302±0.259), and the difference was statistically significant significant among the three groups (P<0.05). The observation group and the positive control group had statistically significant difference (P<0.05), and the negative control group and the observation group had statistically significant difference (P<0.05). After cell transplantation, the exogenous mesenchymal stem cells entered the blood system of rats, which had the conditions of activating the immune system directly; at the same time, they spread to the whole body with the circulation system migration; after transplantation, the rats were dissected at different time points, and the visceral organs, brain tissues and skeletal muscles were observed carefully. Their tissue morphology was basically normal, and no tumor was found. The rats were killed at 8 weeks after cell transplantation, the spleen was dissected and the spleen index was calculated. The results showed that the spleen index of the experimental group (58.7±12.4) was not significantly different from that of the control group (57.3±21.5) (P>0.05). Conclusion? ?Bone marrow mesenchymal stem cells cannot effectively activate lymphocyte cell in vitro, and it does not significantly up-regulate the host's immune status in vivo, and has no obvious tumorigenic performance. It is a good seed cell selection for cell transplantation therapy.
【Key words】 Bone marrow mesenchymal stem cells; Immunogenicity; Cell transplantation
骨髓間質干細胞是存在于骨髓中具有向多個胚層細胞分化能力的干細胞, 在體外能夠大量復制擴增, 既往研究證實, 骨髓間質干細胞可分化表達多種細胞表型。正是因骨髓間質干細胞具備自我復制擴增和向多個胚層細胞分化的特點, 現已廣泛應用于多種疾病的研究[1, 2]。其中一個重要的研究方向就是利用骨髓間質干細胞作為種子細胞來移植治療多種疾病[3]。而種子細胞的免疫原性是移植能否成功的重要因素之一。本研究在體外分離培養(yǎng)來源于大鼠的骨髓間質干細胞, 觀察其生物學特性演變并進行同種異體的細胞移植, 探索骨髓間質干細胞的免疫原性?,F報告如下。
1 材料與方法
1. 1 SD大鼠骨髓間質干細胞的分離和擴增 雄性SD大鼠, 體重約150 g, 處死后用75%的酒精進行消毒, 時間為10 min,
無菌條件下快速解剖分離出雙側后肢股骨, 剪開股骨兩側頭部, 用DMEM-F12培養(yǎng)液沖洗骨髓腔, 收集骨髓沖洗液, 經離心(1200 r/min) 5 min后收集細胞, 含10%胎牛血清的DMEM-F12培養(yǎng)液重懸, 重懸液經100目濾網過濾后接種于培養(yǎng)瓶, 置于37℃、5% CO2培養(yǎng)箱中培養(yǎng), 24 h后更換培養(yǎng)液, 每3~4天更換培養(yǎng)液1次, 待細胞生長融合, 鋪滿培養(yǎng)瓶底部后, 用2.5 g/L的胰蛋白酶消化后按1∶2進行傳代培養(yǎng)。
1. 2 體外淋巴細胞增殖實驗
1. 2. 1 淋巴細胞分離 取健康人外周血約3 ml(肝素抗凝), 用Percoll淋巴細胞分離液分離出淋巴細胞。
1. 2. 2 刺激細胞的制備 取第5代骨髓間質干細胞, 生長至約80%匯合時, 用胰蛋白酶消化, 離心后用含20%小牛血清的1640培養(yǎng)液調整細胞濃度為5×105/ml。
1. 2. 3 單向混合淋巴細胞反應 為了確定骨髓間質干細胞是否具有刺激人的外周血淋巴細胞增殖的作用, 將分離的外周血淋巴細胞和骨髓間質干細胞按1∶1混合, 進行淋巴細胞增殖實驗。設立陽性對照組和陰性對照組, 具體如下:陰性對照組為人外周血淋巴細胞;陽性對照組為人外周血淋巴細胞和植物血細胞凝集素(PHA)(100 mg/L, 20 μl/孔)混合培養(yǎng)組;觀察組為人外周血淋巴細胞和大鼠骨髓間質干細胞混合培養(yǎng)。實驗體系在96孔培養(yǎng)板中進行, 每組設3個復孔, 每孔接種細胞數為1×104個。96孔板接種細胞后置于37℃、5%CO2環(huán)境中培養(yǎng)2 d加入CCK-8 (10 μl/孔), 混勻后孵育4 h后用酶標儀檢測吸光度值。
1. 3 經尾靜脈移植大鼠骨髓間質干細胞 取第五代細胞, 常規(guī)消化離心收集細胞;培養(yǎng)液重懸錐蟲藍染色后鏡下計數細胞后重懸。實驗組大鼠固定后經尾靜脈緩慢注射0.5 ml的細胞懸液, 移植細胞總數約為2×107/只, 對照組大鼠經尾靜脈注入等體積生理鹽水。
1. 4 移植后大鼠脾臟指數及各器官的致瘤性觀察 分別在細胞移植后第4、8、16周處死大鼠, 解剖大體器官, 觀察器官形態(tài)結構有無異常, 是否出現腫瘤。
用于脾臟指數觀察的大鼠選擇細胞移植后第8周大鼠, 沿腹中線剖開腹腔, 剝離脾臟, 盡可能將脾臟上的脂肪完全清除后稱重。計算每只大鼠的脾臟指數。計算公式:脾臟指數 =脾臟重量(mg)/大鼠體重(g)×10。
1. 5 統(tǒng)計學方法 采用SPSS23.0統(tǒng)計學軟件對數據進行處理。計量資料以均數±標準差( x-±s)表示, 采用t檢驗, 多組比較采用方差分析;計數資料以率(%)表示, 采用χ2檢驗。P<0.05表示差異有統(tǒng)計學意義。
2 結果
2. 1 體外培養(yǎng)的大鼠骨髓間質干細胞生物學特性 骨髓腔沖洗液接種培養(yǎng)瓶后可見大多數為懸浮細胞, 培養(yǎng)1 d后部分圓形細胞呈現貼壁生長, 培養(yǎng)2 d后更換培養(yǎng)液, 可見貼壁生長的細胞逐漸向梭形演化, 培養(yǎng)5 d后大部分細胞外觀呈成纖維細胞樣表現。繼續(xù)培養(yǎng)10 d后多數細胞為長梭形, 形成致密生長層。傳代培養(yǎng)后, 細胞生長速度明顯增快, 約3、4 d即可進行傳代培養(yǎng)(圖1)。大鼠骨髓間質干細胞在體外增殖能力強, 反復傳代至10代以后, 仍能良好生長, 且生物學特征無明顯變化。
2. 2 體外淋巴細胞增殖試驗結果 各組在加入CCK-8試劑終止反應后用酶標檢測儀測得的結果顯示:觀察組為(1.515±0.737), 陽性對照組為(1.966±0.421), 陰性對照組為(1.302±0.259), 三組比較差異具有統(tǒng)計學意義(P<0.05), 觀察組與陽性對照組比較差異具有統(tǒng)計學意義(P<0.05), 陰性對照組與觀察組比較差異無統(tǒng)計學意義(P>0.05)。
2. 3 細胞移植后成瘤性觀察 細胞移植后, 外源性骨髓間質干細胞進入大鼠血液系統(tǒng), 具有直接活化免疫系統(tǒng)的條件;同時隨著循環(huán)系統(tǒng)遷移向全身播散;在移植后不同時間點解剖大鼠, 仔細觀察各內臟器官、腦組織和骨骼肌, 組織形態(tài)基本正常, 未發(fā)現腫瘤形成。
2. 4 細胞移植對大鼠脾臟指數的影響 細胞移植后第8周處死大鼠, 解剖分離脾臟, 計算脾臟指數, 結果顯示, 實驗組大鼠脾臟指數(58.7±12.4)與對照組的(57.3±21.5)比較差異無統(tǒng)計學意義(P>0.05)。見表1。
3 討論
干細胞移植已經成為很多難治性疾病有希望的治愈方法之一, 目前已經成為醫(yī)院的研究熱點。外源性干細胞移植能否成功的因素有很多, 其中外源性細胞免疫原性引起的免疫排斥反應和致瘤性是重要的影響因素[4]。因此, 對種子細胞的選擇至少應該具備2個特性:①免疫原性低, 不刺激宿主產生較強的免疫排斥;②不能造成外源性腫瘤發(fā)生。
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[收稿日期:2019-08-09]