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Effect of Danshen Injection with Neural Stem Cells Transplantation in Rats

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Effect of Danshen Injection with Neural Stem Cells Transplantation in Rats ęඈඋ1࣏ۘऺ ৰ 21 Ż ৰ 29 ƛ 20171018 ϦČ Chinese Journal of Tissue Engineering Research October 18, 2017 Vol.21, No.29 www.CRTER.org ऺƣጛۘ %džͼȸ^ȭࠢඓ¾ඊྡྷࢿΉ͏ћ9࿕ǃĐՈƽˑ Ï1 ɏ ᔒ2(1ǧSďS˂ˡŋʒ½ˇˊãKǧSď 300350Z2ǧSḓ̌̈́½ÒlȻãʜDKǧSď 300350) .Ï ɏᔒ. %džͼȸ^ȭࠢඓ¾ඊྡྷࢿΉ͏ћ9࿕ǃĐՈƽˑ[J].ęඈඋ1࣏ۘऺ 2017 21(29):4709-4715šϬƨ DOI:10.3969/j.issn.2095-4344.2017.29.020 ORCID: 0000-0003-3252-1452(Ï) তȷợⓉᪿ ǎ̓K˃K1975 ˝ÛKǧ 9࿕ǃĐ۔džͼȸ^༘Ȍࠢඓ¾ඊྡྷࢿΉ͏ћ̓% SďNK˩ȂKƶƍåK ƶϙÎāÒlȻãΡ~ ඈ¾8 Ụµ ̼ϟūʃ ඗Ș Ě ିǻ:R394.2 (1)࿕ǃĐඈᖟตၠ[ဘͼȸ B:ۅǒɀ¬ĭ ₑ Ƨ ^ (1)ࠢඓƚ෾ǃ᪈ (1)%džͼȸ^༘Ȍࠢ Dzʃ᪊ DMEM ȕͧ^ ඓ¾ඊྡྷᖟตၠ[ဘࢿ তේǻ:2095-4344 Ə 9 ࿕ ۔̓ SD (2)%džͼȸ^ඈွဘͼȸ%dž ࿕ (2017)29-04709-07۔ǃ Đ µ (2)Morris ˈẻǟ Ήdz࿁ỞẋƯẟ̓ ͼȸ^ Ƨ ǃĐľ࿕ඈඋॅᢪ௤ǎ ɘ4ěY2017-05-03 ǒɀ (3)¾ඊྡྷඈᖟตၠ[ဘͼȸ ࿕ඈඋ҉ˊඈ ϣ⑃ּ͟ᖏՁ 43 Ոᜬ(3) CM-Dil ʃᩴՈࠢඓ¾ඊྡྷ˸^ උ ƚ ᢆ ȓ Ẃ (4) ༘ Ȍ ඈ ᖟ ต ၠ [ ဘ ͼ ȸ (4)RT-PCR ǎ (2)ΝΈₑƧ9࿕ǃĐ CM-Dil ʃᩴࠢඓ¾ඊྡྷඊྡྷ˸ Ոࠢඓ£࿁4۔Western blot ̼ µƧ̓ ^ՈȐrွဘͼȸ%džͼȸ^4 ϟ4 L₎5 ࠢඓ¾ඊྡྷࢿΉ͏ћ࿕ǃĐࠢඓ¾ඊྡྷ«P࢑dzȕࠢඓ̯ ĚՈ¾ඊྡྷ ᪩ඊྡྷẜdzÑẟᜐႮuŰ,ʐႮ uමť  ̶͠࢑ࠢඓüƄÑΝΈ9࿕ǃĐ4IJ«ϵzࠢඓ¾ඊྡྷńࢿΉẋ࣏ƌϏƅ└ ͢ࢿΉՈ͏ћ άȘKǛ4rṇ̓└:4໐%džͼȸ^dzỞẋʺȪࠢඓ¾ඊྡྷƯẟȒ້ࢿΉՈћά4 džՈ£ά%dždzÑ෗ᢧͱղǃĐ ęû̺ҋǑà ΝΈǺǶɳ ᫇ᅆச࿆ĿͱÏ᫦ Ǹ̲ۘऺᜬ4%% džń͏ћࠢඓɳ҂҉5☦ћάºጛ4%džȴǚ^dzºጛȴʌࠢඓ¾ඊྡྷՈʺȪϏɳ ΝΈࠢඓ¾ඊྡྷࢿΉՈ ƌϏ4 ʼᡅ ྐë%džȴǚ^dzºጛȴʌࠢඓ¾ඊྡྷՈʺȪϏɳ ΝΈࠢඓ¾ඊྡྷࢿΉՈƌϏ4 9࿕ǃĐՈƽˑ4۔ՈȆᩬ%džͼȸ^ȭࠢඓ¾ඊྡྷᖟตၠ[ဘࢿΉ͏ћֲ̓ 4 ඈ¾8 tₑƧ^Ə9࿕ǃеƧ Ⱥņµt£Ո 80 Ǯ╓ƶ:۔̓ 5ͩọǚ 87 ǮtÀ SD ࿕ǃĐඈᖟตၠ[ဘͼȸ DMEM ȕͧ^ 1 Ƶ/d%džͼȸ^ඈွဘͼȸ%džͼȸ^ 1 Ƶ/d¾ඊྡྷඈᖟ ตၠ[ဘͼȸ CM-Dil ʃᩴՈࠢඓ¾ඊྡྷ˸^ 1 Ƶ/d༘Ȍඈᖟตၠ[ဘͼȸ CM-Dil ʃᩴࠢඓ¾ඊྡྷඊྡྷ˸ ^ՈȐrွဘͼȸ%džͼȸ^ 1 Ƶ/d ȈඈẢන¾8 3 d4¾8Ȓ 3 d ǎ 1 2 3 4 ɬᜐࠢඓƚ෾ǃ᪈ ¾8 21¤28 d ẟᜐ&ƛ 7 d Ո Morris ˈẻǟǒɀ¾8Ȓ 4 ɬẟᜐ࿕ඈඋ҉ˊඈඋƚᢆȓ3ࠢඓ¾ඊྡྷƌϏ ^ Oᢆȓ3RT-PCR ǎ Western blot ̼ϟ4 ඗Ș^඗᩾ ࠢඓƚ෾ǃ᪈ ༘Ȍඈࠢඓƚ෾ǃ᪈ ϧඌĄz͢Ņ 3 ඈ(P<0.05) Morris ˈẻǟǒɀ z͢ 3 ඈ(P < 0.05 z P < 0.01) ृᱎ¿ǴƵϔǎńֲʃᬥ└äڱȌඈ¾8Ȓ 3¤5 d Ո¿ţȈûrⒸº༘ ·Სࡿ^ʇᲡࡿՂ ɨţʌz͢ 3 ඈ(P < 0.05 z P < 0.01) ࿕ඈඋ҉ˊඈඋƚᢆȓ࿕ǃĐඈdzᢅ࿕ǃ Đľඈඋाβº %džͼȸ^ඈ3¾ඊྡྷඈ࿕ǃĐľඈඋाβṇɃ ༘Ȍඈ࿕ǃĐľඈඋाβό:4͍ ࠢඓ¾ඊྡྷƌϏ^ O༘Ȍඈ CM-Dil ʃᩴ⓷ɳඊྡྷϔ̶z¾ඊྡྷඈRT-PCR ǎ Western blot ̼ϟ Ȍඈ࿕ඈඋॅᢪ௤ǎϣ⑃ּ͟ᖏՁ 43 ᜬẂʌz͢ 3 ඈ(P < 0.05) %džͼȸ^ඈ3¾ඊྡྷඈʌz࿕ǃĐ༘ ࿕ǃĐľ࿕ඈඋॅ۔ඈ(P < 0.05)඗Șᜬ%džͼȸ^༘Ȍࠢඓ¾ඊྡྷᖟตၠ[ဘࢿΉdz࿁ỞẋƯẟ̓ Ոࠢඓ£࿁4۔ᢪ௤ǎϣ⑃ּ͟ᖏՁ 43 ՈᜬẂ ΝΈₑƧ9࿕ǃеƧ̓ ͟⏲᪑ ඊྡྷࠢඓ¾ඊྡྷ%džͼȸ^ࢿΉ9࿕ǃĐॅᢪ௤ϣ⑃ּ͟ᖏՁ 43ᖟตၠ[ဘ¾ 'L᪑ ¾ඊྡྷࠢඓ¾ඊྡྷልሳඊྡྷࢿΉඈඋ1࣏ Ȗₕᰈ­ (ιNůϣᜐₑͥΟ͟-ֲ(2013KG134ͅ Effect of Danshen injection with neural stem cells transplantation in rats with Liu Xiao, Attending physician, Department of craniocerebral injury Brain, Xianshuigu Hospital of 1 2 1 2 Jinnan District, Tianjin Liu Xiao , Wang Hu ( Department of Brain, Xianshuigu Hospital of Jinnan District, Tianjin 300350, China; Fifth 300350, China Ward, Department of Neurosurgery, Tianjin Huanhu Hospital, Tianjin 300350, China) ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 4 7 0 9 Ï ਍. %džͼȸ^ȭࠢඓ¾ඊྡྷࢿΉ͏ћ9࿕ǃĐՈƽˑ www.CRTER.org Abstract BACKGROUND: Danshen injection can dramatically enhance the proliferation and survival of neural stem cells (NSCs) after transplantation. OBJECTIVE: To investigate the effect of Danshen injection with NSCs transplantation in rats with craniocerebral injury. METHODS: Eighty-seven adult Sprague-Dawley rats were used to build severe traumatic brain injury models. After successfully modeling, the model rats were randomized into craniocerebral injury group (the subarachnoid injection of DMEM medium; n=20), Danshen injection group (the intraperitoneal injection of Danshen injection; n=20), and NSCs group (the subarachnoid injection of CM-Dil-labeled NSCs suspension; n=20) and combined group (intraperitoneal injection of Danshen injection plus the subarachnoid injection of CM-Dil-labeled NSCs suspension; n=20). The injection in each group was performed once a day for 3 consecutive days. The modified neurological severity scores were tested after 3 days, 1, 2, 3 and 4 weeks of the transplantation. The Morris water maze test was performed at 2128 days of transplantation. NSCs survival and distribution were detected by fluorescence microscope and the pathologic changes of brain tissues were observed at 4 weeks after intervention. The expressions of synaptophysin and growth associated protein 43 at gene and protein levels were tested in the brain injury zone by RT-PCR and western blot assay respectively. RESULTS AND CONCLUSION: The modified neurological severity scores in the combined group were significantly lower than those in the other groups (P < 0.05). In the Morris water maze test, the average escape latencies in the combined group was significantly shortened in the combined group after 35 days of transplantation in comparison with the other three groups (P < 0.05 or P < 0.01); the frequency of passing through the platform and the percentage of the swimming distance at the target quadrant in the total swimming distance were significantly higher in the combined group than in the other groups (P < 0.05 or P < 0.01). The hematoxylin-eosin staining results showed the tissue voids in the Danshen injection and NSCs group were relatively smaller than those in the craniocerebral injury group, and the voids almost disappeared in the combined group. There were more CM-Dil positive cells in the combined group than the NSCs group. RT-PCR and western blot assay showed that the gene and protein expression levels of synaptophysin and growth associated protein 43 in the combined group were the highest followed by NSCs and Danshen injection groups, and the lowest expression levels were observed in the craniocerebral injury group. In summary, the Danshen injection combined with the NSCs transplantation can improve the neurological function of rats with severe craniocerebral injury, which might function by increasing the expression levels of synaptophysin and growth associated protein 43. Subject headings: Stem Cells; Neural Stem Cells; Drugs, Chinese Herbal; Cell Transplantation Funding: the Key Project of Health Industry in Tianjin, No. 2013KG134 Cite this article: Liu X, Wang H. Effect of Danshen injection with neural stem cells transplantation in rats with craniocerebral injury. Zhongguo Zuzhi Gongcheng Yanjiu. 2017;21(29):4709-4715. 0 šᣄ Introduction 1.2 ðϏΛıļ ǒɀz2015À1ƄႷ2016À10Ƅńͅ 9࿕ǃĐȏɋʐǕ҉ɋǽʌÂɌɴϦỔÀZŋՈᱏ ιĿ࢕̓ƚ¬ĭǒɀǘǀt4 Ã[1-3]4ɱ੥ĿƚА])ȴϦ,ՈᢧΟ5˄ IJ«ֲQę 1.3 ƨ 87Ǯ Ŀᯬₓ൪۔᪩҉Ոȏɋ¹ϢʌẂ40%2Ƿ[4]4ࠢඓ¾ඊྡྷ«P࢑'ᡅ ǒɀ¬ĭ2ƄܸɑătÀSD̓ ƌńzȞࠢඓிඣՈ¾ඊྡྷ ᪩ඊྡྷ̠zƦ Ěźɍ 300 g ▐█ȈŎ ϵͅιĿ࢕̓ƚ¬ĭǒɀǘȴƇ ϣ dzỞẋ̶࢑5śʺȪ Ě[5]4᪩ିඊྡྷ>ᝯËͯàϬz͏ћ ᩼dz᪅SCXK(ι)2013-0003 ǒɀ¬ĭūϬ᩼dz᪅ǻ zǘÕ25 ?3īò50%۔᪼Ξ࿎ʇǃĐ3໅ÀҸɊ਍̶࢑ࠢඓ҂҉ՈÂǚǣrPǎ࣏ SYXK(ι)2013-00764¤ƅ̓ òՈẟʉ[6-10]4ۘऺº߾ ͱļɳՈࠢඓ¾ඊྡྷńࠢඓǚ̩ 80%3SPFɳʟ[ႮϵĢˈʐ₋4 ǕƉ؄ǽ͢ₑᡅՈňϬ[11-16] ̶᪼ۘऺỞẋࠢඓ¾ඊྡྷ 'ᡅ᪙FǎÖྴᖏՁ›ϵ๒ęSigma͘Ǽᯱẟ☦5 ՈࢿΉKǚǣrṇΙάȘ[17-20]4Ϣ໐ࢿΉẋ࣏ǽ&ƅ└Ո ඊྡྷȕͧੵᯱ\ႮHeraeus Sepatech ͘ǼDMEMȕͧ ࠢඓ¾ඊྡྷƌϏ਍̶᪼Ո⒲LKǽ࣏̓òZƽˑr᪩ћͩ Ȗ3ྒğᜄ±ʐPBSţᯱႮHycloneTrizol᪙F֖ᯱႮ ՈάȘ[21-22]4%dž«ęՈČඣሳnj7P ü͢ȯƅ̶࢑„ InvitrogenBCAᖏՁϧòϟ᪙᪙F֖(ʺƆƧ)35×SDSᖏ ਍Ϗɳĭᯬ໐Ǜ4Ŀƚۘऺ້Ո☖ؔ4%dždzÑ෗ᢧͱղ ՁZʳ෗Ξ^320TBS෗Ξ^਍ţᯱ\zśņtϣĭ‼ ǃĐ ęû̺ҋǑà ΝΈǺǶɳ ᫇ᅆச࿆ĿͱÏ᫦ Ǹ ඊྡྷ̠ˊ਍ẋ࣏ūϬՈ᱉ά1ňǴȴƇႮᄾ+ʌZ#᯼ ̷̶¦ऺᜬ%džń͏ћࠢඓɳ҂҉5☦ћάºጛ[23-25]4Ц ƅ└͘Ǽϣ⑃ּ͟ᖏՁ433ॅᢪ௤Pû&̀û̲ۘ ͧ͠Ո«%džȴǚ^dzѺጛȴʌࠢඓ¾ඊྡྷՈʺȪϏɳ ╊ûĿ xû&ṧʵẋʻĚĭ›(HRP)ʃᩴՈʥ๎û̶̀ û¦ûĿ GAPDH۔ΝΈࠢඓ¾ඊྡྷࢿΉՈƌϏ[26-27]4üǸ ǒɀdńỞẋ༘Ȍ ̷╊ûĿ GAPDHPû&ř̷╊Ƀ Ո̶̷╊ûĿ ¤ƅûĿ۔9࿕ǃĐՈࠢ Ոxû&HRPʃᩴՈʥ๎ûɃ۔džͼȸ^ʐࠢඓ¾ඊྡྷ ẟ໐Ȇᩬ͢ȭ̓% ඓǚ̩ňϬ Ñƛ&9࿕ǃĐ͏ћȴƇȋℸʐΓť4 ῁ᯱzᅂăϣĭäƫ(ǩ*)ƅ└͘Ǽ%džͼȸ^ᯱ\Ⴎ ÷)ŋʐሳ^ཥéƅ└͘Ǽ4 1njʐ5ͩMaterials and methods 1.4 ôκǒƽ ۔ඈ ǚ87ǮtÀSD̓ µƧǎǒɀ۔Éα ╓ƶȭ+¬ĭǒɀ4 1.4.1 ņএ̓ 1.1 4710 P.O. 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