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

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Effect of Danshen injection with neural stem cells transplantation in rats with Liu Xiao, Attending craniocerebral injury physician, Department of 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

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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.

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¿Ẕ¬Ḭẽǎȯâ۔ţʵǒₑƧ9࿕ǃ ிඣּẢȉՈʨʻƶ,ǳȐǹᩴơ̓۔ňₑƧ^Ə9࿕ǃĐµƧ4¤ƅ̓: Đę┉ʃβ₋Ϭ^Ə9࿕ǃĐÖඝ^͢ʌƏΞϧ Ȁņₑ ǴȈûƛ਍4 ẟᜐǒ۔/╓ƶọǚ6Ǯ̓ Ƨ^Ə9࿕ǃĐµƧ4 ǎĹხᜐǒɀºȈඈ &6൫1൫ ɀ ᪩ǒɀ◄ẟᜐ7 d(ɣͅZ[ŌȈẟᜐ1Ƶ ɣƵᩱඇ2 ՈͫᢍʐẔ¬࿁Ⱥ7۔ỤµȒʵǒ̓ ΢4ƽ͔¿ǴZ۔ń਀ͱǳÑǷϏ¬2൫ Ϗ¬ₓλɅ3൫ Ϗ¬ₓ ἑ)4ᩱඇ7QŊ̴Ⱥ¤ƅǆ^ǒɀՈ̓ ºλɅZẔ¬͍᫇4൫ ྐᾬ΢ń਀árǳÑǷƆ¬ ɈН2.0 3.0 min ū7c˕ǒɀźΡʐǒɀɳʟ4ǒɀẟ ˈ͑ˈ˴ºǸ͑ˈͥྐȭ۔Ĳ]࿁ঝএ5൫ ྐᾬᢪáȒ]ǳ຿Ɔ ĲͣƅསĿҀҟ ᜐr╓ƶōǚP͑ˈͥ Ⱥ̓ Ո͑ˈͥPႸŌϧᩴơǒɀ¬ĭº۔ú෭Ǒà6൫ ȭçŁ>ɬ\Ǒà4ǒɀÑ4൫ʐ5൫ǃĐ ÂūȐƵǒɀ̓ Ⴎ'ʀȼČ▂ এŷ ͑ˈͥ͑ˈȒ4Ẃ¿ǴՈrⒸⒸ╘(ǸŷệỼȈûƛ)4Ξ۔ň&൷͑ʃβ4ńņµẋ࣏ᆩϦɴ̓ àȺ7šႷ ȭ͢ǒ9Ƀ¬ĭ☦อȼʻẟᜐĆε4ń᪩ẋ࣏ƟŖȏ2Ǯ Ș͑ˈ90 s7Ȓǒɀ¬ĭ¹Ʀ4ֲʃŌͥ ᩴơ&90 s4Ⱥɣͅ4ƵՈệỼȈ80Ǯ4Ⱥਟọ ¿Ǵ ໐ƨƵrⒸⒸ╘&۔̓£ņµȒ12 hȏ5Ǯ Ụµt ۔̓ &4ඈ࿕ǃĐඈ3%ǆͼȸ^ ûƛՈਜ਼ƫ¿ţȨň&ẝPͅՈƚLtත4 ƶ╓۔ƌϏ[ǡՈ80Ǯ̓ ඈ3¾ඊྡྷඈǎ༘Ȍඈ ɣඈ20Ǯ4 1.5.3 ¾8Ȓ4ɬỞẋᆓƤஂ ö൦ɏᄶᢆȓ҉ˊƚΝǜ ϔ 3ǮÂǚϦ9࿕ǃĐඈඋ ϬĿࢳ۔ȏ̠̓⁍ٯࠢඓ¾ඊྡྷՈĿ̲ȕͧǎCM-Dilʃᩴ Ⱥᯱ\Ⴎ ɣඈ 1.4.2 ęĿƚ࢕ƚ┦ՈΧƌƣÏࠢඓ¾ඊྡྷาz60 ?ʞÕ 10%϶ Ėǎඈඋ:̣9࿕ඈඋ ċūϬϵĺϧò4ʌ ˈ ੵ 30 sͱẟᜐࠢඓ¾ඊྡྷȷợ̩ᆓ4ȺᙑĚ7ȒՈ ϧòՈE⁋ň࿵ˈF Ổ¼࿵ƿඈඋųՈˈ 4͹Ⱥඈ ᗥՈồFx϶ᆳồڷࠢඓ¾ඊྡྷẉ҂Ḱ͑EP੥Â¤͑DMEMȕͧȖ ÂZ̶ උųาz^ŢzE⁋ ǌŢz Ƶₑ̩ɃȏȽ·ඊྡྷū͢ţĄ O ╓ȒࡿȏŏZ±^4 Ñx϶ᆳŻǆϦඈඋųՈE⁋ ╓Ȓẟᜐ$ᗥĉǧ4Ⱥ Ⱥ̩ᆓȒՈඊྡྷϬDMEMǀ͔ȕͧȖ᫇ϘඊྡྷϧòႷ ĉǧΙՈᗥųĖǎz ċƶZ t5 8 µmƞᑈċ4Ⱥᑈ 1×109 L14ȕͧ3ÏȒ ྴᖏՁ›Ţ^4Ě2.0 3.0 minȒඌ ċΗ¿ ͹᯸4ṁɿċZ 45 ?ʞÕ΄¾4࿵ᗥȒ ẟᜐ Ƕ4ĚࡿȏŏZ±ₑ˸ඊྡྷ4᫇ϘඊྡྷϧòႷ1×109 L1 ᆓƤஂ ö൦ɏᄶͩɏᄶ4ɏᄶȒՈ ċඓ൳E⁋࿵ˈ ͹ ȕͧ7 d4ඓẋ3ƵΫP7ȒȺࠢඓ¾ඊྡྷϧò᫇Ϙ4 ඓx϶ᆳū ċồ4Ⱥ>ồՈ ċƠZ¤ţ̓ʍྺ L1 ╓Ȓȕඊྡྷ˸^ஂܲ¤͑5 µL CM-DiLẟᜐ ֚Z֚ɿċȵĖ4Ǒʍྺ¾©Ȓ ራ̵ºǺ␠[ᢆȓȈඈ 108×8 9࿕ǃĐ҉ˊƚǜĚ4۔̓ 30 min ʃඊྡྷʃᩴ╓ȒาzȕͧੵʃᩴƩĚ25ֲ ࡿȏŏZ± PBSₑ˸ ₑ̩ʃᩴ2Ƶ ºǺ␠[ᢆȓඊ 1.5.4 ራ̵ºǺ␠ᢆȓCM-DilʃᩴՈࠢඓ¾ඊྡྷƌϏǎ 9࿕ǃĐľǻඈඋ :ň۔̓▊₋ ȎΡ ¾84ɬȒ ʃᩴՈ̑Ρ4␠[ǳᢅࠢඓ¾ඊྡྷʃᩴºɴϦѐᄷՈìྡྷ 2ᆳȖȶ x࿖Ȗ ċÂẟᜐ4’ 6 9࿕ǃĐඈඋΜΧ۔̓ ൦ᄶZPᄶţĄ ̣Ϭ4 ࿕ǃĐȒ24 h ࿕ǃĐඈºȑɜ̓ƈ̠Ñ1 µL/minՈ ˞ɏᄶ4¾©Ȓ ºǺ␠[ᢆȓr ╓ƶọǚɣŬΣΧ ͼȸợòᖟตၠ[ဘͼȸ10 µLඊྡྷȕͧ^ 1Ƶ/d%ǆ ċՈ3ᢊₒÂZȭ7ẟᜐı+ᩴơȳ᪂ɣŬ+ċՈʇ ͼȸ^ඈွဘͼȸ%ǆͼȸ^0.5 g/kg 1Ƶ/d¾ඊྡྷඈ ò&1 ɣŬ+ċọǚP̵òͥ -ϬACDsee 5.0ḳ ᖟตၠ[ဘ෗ήͼ͑10 µLࠢඓ¾ඊྡྷ˸^(3×106 L1) âܲǎ͢ŬʃȨ4ńĚʻ╓ƶọǚ8ᢊₒ ᩥਜ਼9࿕ǃ 1Ƶ/d༘Ȍ͏ћඈᖟตၠ[ဘ෗ήͼ͑10 µLࠢඓ¾ඊྡྷ ĐඈඋՈࠢඓ¾ඊྡྷʃᩴɋ4 ࿕ĐľɬĐॅᢪ௤ǎϣ⑃ּ͟ᖏ۔L1)ՈȐrွဘͼȸ%ǆͼȸ^0.5 g/(kg·d) 1.5.5 RT-PCR̼ϟ̓ 106×3)^˸ Ȑ۔1Ƶ/d ȈඈẢන¾83 d4 Ձ43ȖüḰơՈᜬẂˈ¿ǜĚ ¾84ɬȒ ǚ4ඈ̓ 1.5 ƶϙʬ+ŷ/ PᾬĹՈ9࿕ǃĐඈඋ ₋ϬRT-PCR̼ͩϟ9࿕ǃĐඈ උϣ⑃ּ͟ᖏՁ43ʐॅᢪ௤ȖüּȭᜬẂˈ¿4ɣ̼ 8Ȓ3 dǎ1 2 3 4ɬᜐ¬ĭࠢඓƚ෾ǃ᪈¾ 1.5.1 ϟ᪙ ࠢඓƚ෾ǃNSS᪈ ǆ໇5 :᪈ Ŀிẟᜐ̱ň ϟₑ̩3Ƶ4ỞẋOligo 7.36 Demoḳâ᪂ᩥšĭ ॅᢪ௤ /z¾8Ȓ3 dʐ1 4ɬ(ɣɬżȒ1 d)ϟǎ ͢᪈ ʃβ Zäšĭ5’-CAG CCG TGT TCG CTT TCA T-3’ [ä &0 \ࠢඓிඣǃĐĿǍ 1 ¯ƫȭƓQîƅ šĭ5’-CCA CCC GTG GGA TCT TCA T-3’ Íʺċɉ ȕ¯ƫȭƓ 650 bpϣ⑃ּ͟ᖏՁ43 šĭÛZä5’-AGG AAA ȕ¯ƫȭƓḰĤ3 ºՈĤָ╠ݑ2 ȪǾ4 ]࿁ႮǕᜐᰴ ͛᪊͍4 GGA GAG AAG GCA GG-3’[ä5’-GCA GGA GAG 1.5.2 ¾8Ȓ21 28 dᜐMorrisˈẻǟǒɀ Ŋ̴ȕˈẻ ACA GGG TTC AG-3’ Íʺċɉ⑃ò778 bpͱǆ ǟញา¤͑̓൪1 kgΒ୍ ╓Ȓ¤ˈū͢ʌò᱉ẋƽ͔ GAPDH Zäšĭ5’-GTG CTG AGT ATG TCG TGG ¿Ǵ̓൪1 cm4╓ȒńˈẻǟՈ˴˝Zʃᩴ4͑ˈͥ(ʵ A-3’ [äšĭ5’-CAC AGT CTT CTG AGT GGC A-3’ ǒᡃśěՈ5Ĺ /ʃᩴ1 2 3 4)Ɖ+4Ϧˈͥ Íʺċɉ121 bp4ȴǚඊྡྷRNAÂǑḰơ&cDNA ₋Ϭ Ȍͩ ÑGAPDHȖüň&ͱǆ ϟǎϣ⑃ּ͟ᖏ &4ᬥ└ÂȺƽ͔¿Ǵาzě 2∆∆Ct ȺˈẻǟՈˈ˴ţ਍ ᬥ└ࡿੵ˝15 cmՈŌ5Ṇ̃ǒɀ4ẻǟZ5ƽา^Ϲ࿕ Ձ43ʐॅᢪ௤mRNAּȭᜬẂˈ¿4

ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 4711

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ࠢඓ£࿁ţńỔ¼ʮ̩Ƿ4ń¾ඊྡྷࢿΉ3 d7Ȓ۔̓ ּ⑃࿕ĐľɬĐॅᢪ௤ǎϣ۔Western blot̼ϟ̓ 1.5.6 ͟ᖏՁ43ᖏՁՈᜬẂˈ¿ǜĚ ¾84ɬȒ ȴǚÂϟǎ ༘Ȍඈࠢඓƚ෾ǃ᪈ ºጛĺz͢ÂȈඈ(P < 0.05) %ǆ º 9࿕ǃĐඈඋᖏՁϧò Ⱥඊྡྷឆᢧ7ÂȺ7ࣄ₎ႷּȐ ͼȸ^ඈʐ¾ඊྡྷඈ¾8Ȓ2 3 4ɬՈࠢඓ෾ǃ᪈ ՈᖏՁϧò 100 ?2͌5 min ūᖏՁǜɳ4ɣඈǚᖏՁ ጛĺz࿕ǃĐඈ(P < 0.05) ᢅᜬ14 µg 10%SDS-PAGEωྺϹ·(70 V 30 min100 V 2.3 MorrisŋɩɽôκBˌ Morrisˈẻǟǒɀ ╓؄ 40 ¼ţệỼȈûƛȈûrⒸỔ¿۔min)Ḱၠ(200 mA 3 h)ႷPVDFၠ5%࿵࿆ğΒǘ ǒɀrⒸՈł⑃ Ȉඈ̓ 90 (ǒɀŌϧՈPxͅ Ȉඈ7Ⓒ\ºጛ:Ŏ(P > 0.05ڱÕȵ⒱2 h(z4 ?ẋ̸)Pû1a500 ǘÕƩྲྀ2 h(z4 ? ෭ ẋ̸)TPBSΫP3Ƶ PBSΫP1Ƶ7ȒẟᜐᖏՁ̹ѯ 3 5 dr ༘Ȍඈ¿ţȈûrⒸṇ%ǆͼȸ^ඈǎࠢඓ¾ඊ (P < 0.01)ڱP < 0.05) ṇ࿕ǃĐඈº෭)ڱȌ ϟǎȈ ྡྷࢿΉඈ෭ ŴẽՈǑà ı+ᩴơ7ȒẟᜐḳâՈĚʻ ඈ9࿕ǃĐඈඋϣ⑃ּ͟ᖏՁ43ʐॅᢪ௤ᖏՁּȭᜬẂ ͙Sͅr ȈඈⒸ:ŎṇɃ ᢅᜬ24 Ȍඈृᱎ¿ǴƵϔǎńֲʃᬥ└ä·Სࡿ^ʇᲡࡿ༘ 4¿ˈ 1.6 Ȍα͸ǫʭ ǒɀ¤ƅϔǒᰈՈඈⒸɨṇ₋Ϭř Ղ ɨţʌz%ǆͼȸ^ඈǎ¾ඊྡྷඈ(P < 0.05) ºʌ _ ü௤5:(One-way ANOVA)࣏Û Ȍ ǒɀ඗ȘÑx±sᜬ z࿕ǃĐඈ(P < 0.01) ᢅᜬ34 ࿕۔̛Aʬ+DŨ͸ȷ) ࿕ǃĐඈ̓ ߾ ÂᢈǎP < 0.05&:Ŏƅºጛɳ͛54 2.4 Ųʱj ඈඋѓǺ ǃĐľՈाβṇ&º ඊྡྷ7ⒸՈⒸ╝ʺ̓ 2඗ȘResults ࠢඓඊྡྷՈྡྷĿյ෭ ࠢඓඊྡྷϦɴūȏ͝ᢧ^9࿕ǃ ÒlȼˍFĝ́Ƨʬ+ ȕͧ1 dȒ ࠢඓ¾ඊྡྷʺ Đඈּɨ %ǆͼȸ^ඈǎ¾ඊྡྷඈ࿕ǃĐľඈඋाβṇ 2.1 ̶3ṇɃ3Ʈź]ᢈ Ƀᾬ ඊྡྷþ᯸˝ȕͧ5 dȒ Ƀ ūȏ͝ᢧՈࠢඓඊྡྷṇɅ ໐༘Ȍඈ࿕ǃĐľඈඋा ࠢඓ¾ඊྡྷʺ̶ &ṇ̓3ƮźᢈՈˇƮ ᢅĚ1A4̹ βό:4͍ ᢅĚ24 ѯራ̵ɏᄶº߾ ࠢඓ¾ඊྡྷˇɌCM-DilƆ⓷ɳᜬẂ4ࠢ 2.5 ÒlȼˍFáͻΛǫ_ē ࠢඓ¾ඊྡྷࢿΉ4ɬ7 ඓ¾ඊྡྷՈʃᩴ CM-DilʃᩴՈࠢඓ¾ඊྡྷńራ̵ºǺ␠ ȒńCM-Dil⓷ɳඊྡྷ5☦ ༘ȌඈՈ(26.37±3.41)%ºጛʌ Ɍ൦ᄶራ̵ ᢅĚ1B4ۘऺ᪅ǒ CM-DiIʃᩴȒራ̵ń z¾ඊྡྷඈ(12.67±2.07)%3࿕ǃĐඈ(0)ǎ%ǆͼȸ^ඈ] ྡྷͱᜬẂࣷǎ ⓷ɳʃᩴɋẂ97%ÑZ ʃᩴඊྡྷƮɍᄳ (0) ᢅĚ34 Ι ࿁ƅάᢆȓඊྡྷńĿ̲Ո᪵Ȱ Ě̑Ρ4 ţệỼȈû¿۔ᜬ 2 %ǆͼȸ^ȭࠢඓ¾ඊྡྷࢿΉ͏ћ9࿕ǃĐ̓ _ Òl͸4ƍͯǫϗ!Bˌ ºϘࠢඓƚ£࿁╠ݑ 2.2 ƛՈƽˑ (x±s s) ǒɀƛ׏ǡ ╓؄ඊྡྷࢿΉ¾8ȒrⒸՈł⑃ Ȉඈ ᪈ Table 2 Effect of Danshen injection with neural stem cells transplantation on the average latency of rats with craniocerebral A B injury

ˈẻǟ ࿕ǃĐඈ %ǆͼȸ^ඈ ¾ඊྡྷඈ ༘Ȍඈ ǒɀrⒸ

1 d 40.28±2.21 38.33±1.54 39.48±1.81 37.27±2.97 2 d 35.52±2.03 30.14±1.72 31.35±1.45 29.13±1.41 3 d 25.71±1.79 15.56±0.78 16.54±1.61 8.58±0.55ab 4 d 19.23±1.67 9.78±0.71 10.23±0.87 5.33±0.12ab Ě 1 ࠢඓ¾ඊྡྷՈℸǎǎʃᩴ ab 5 d 15.66±1.72 5.39±0.52 5.27±0.64 3.45±0.25 Figure 1 Identification and labeling of neural stem cells 6 d 11.90±0.66 5.12±0.33 5.15±0.22 2.92±0.43

ĚͼĚ A &ࠢඓ¾ඊྡྷ Nestin ̹ѯራ̵Ěƚɏᄶ⓷ɳ(×20)B 7 d 10.26±0.57 5.40±0.25 5.01±0.30 2.77±0.61 & CM-Dil ʃᩴՈࠢඓ¾ඊྡྷ(×200)4 ᜬ ͼ^%ǆͼȸ^ඈǎ¾ඊྡྷඈɨṇ aP < 0.05^࿕ǃĐඈɨṇ bP < 0.014

ࠢඓƚ෾ǃ᪈۔ᜬ 1 %ǆͼȸ^ȭࠢඓ¾ඊྡྷࢿΉ͏ћ9࿕ǃĐ̓ _ ɨՈɨ Ոƽˑ (x±s) ᜬ 3 Ȉඈृᱎ¿ǴƵϔǎńֲʃᬥ└ä·Სࡿ^ʇᲡࡿՂ _ T able 1 Effect of Danshen injection with neural stem cells ṇ (x±s) t ransplantation on modified neurological severity scores of rats with Table 3 The percentage of the swimming distance at the target c raniocerebral injury quadrant in the total swimming distance

(%)ɨ r Ⓒ ࿕ǃĐඈ %ǆͼȸ^ඈ ¾ඊྡྷඈ ༘Ȍඈ ඈ/ ृᱎ¿ǴƵϔ ńֲʃᬥ└ä·Სࡿ^ʇᲡࡿՂ

¾ 8Ȓ 3 d 2.56±0.85 2.39±0.77 2.36±0.71 1.91±0.61a ࿕ǃĐඈ 3.28±0.45 22.13±4.20 ¾ 8Ȓ 1 ɬ 2.47±0.92 2.23±0.61 2.19±0.66 1.70±0.52a %ǆͼȸ^ඈ 5.69±0.62 28.36±5.12 ¾8Ȓ 2 ɬ 2.39±0.59 1.84±0.82b 1.80±0.78b 1.58±0.50a ¾ඊྡྷඈ 5.88±0.64 29.04±4.10 b b a ab ab ¾8Ȓ 3 ɬ 2.16±0.56 1.49±0.41 1.46±0.52 1.24±0.44 ༘Ȍඈ 8.66±0.71 50.52±5.27 b b a ¾ 8Ȓ 4 ɬ 1.84±0.63 1.30±0.40 1.28±0.38 0.82±0.43 a b ᜬ ͼ^%ǆͼȸ^ඈǎ¾ඊྡྷඈɨṇ P < 0.05^࿕ǃĐඈɨṇ P < 0.014 ᜬ ͼ^͢Ņ 3 ඈɨṇ aP < 0.05^࿕ǃĐඈɨṇ bP < 0.054

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A B C D

(9࿕ඈඋᢆȓ҉ˊƚΝǜ(ᆓƤஂö൦ɏᄶ ×200۔Ě 2 Ȉඈ̓ Figure 2 The pathological changes in the brain injury zone of rats (hematoxylin-eosin staining, ×200) ʺ╝࿕ඈඋѓǺ ǃĐľՈाβṇ&º ඊྡྷ7ⒸՈⒸ۔/&࿕ǃĐඈ3%ǆͼȸ^ඈ3¾ඊྡྷඈ3༘Ȍඈ ࿕ǃĐඈ̓ ĚͼĚ AD ࠢඓඊྡྷՈྡྷĿյ෭ ࠢඓඊྡྷϦɴūȏ͝ᢧ^9࿕ǃĐඈּɨ %ǆͼȸ^ඈǎ¾ඊྡྷඈ࿕ǃĐľඈඋाβṇɃ ūȏ͝ᢧՈࠢඓඊ ̓ ྡྷṇɅ ໐༘Ȍඈ࿕ǃĐľඈඋाβό:4͍4

A B C D

(9࿕ඈඋ CM-Dil ʃᩴࠢඓ¾ඊྡྷƌϏ̑Ρ(×200۔Ě 3 Ȉඈ̓ Figure 3 The survival of CM-Dil-labeled neural stem cells in the rat brain (×200) ĚͼĚ AD /&࿕ǃĐඈ3%ǆͼȸ^ඈ3¾ඊྡྷඈ3༘Ȍඈ ༘Ȍඈǳᢅ̓ₓ CM-Dil ⓷ɳඊྡྷ ¾ඊྡྷඈƵ7 ࿕ǃĐඈǎ%ǆͼ ȸ^ඈƦᢅ CM-Dil ⓷ɳඊྡྷ4

9࿕ඈඋॅᢪ௤ǎ۔࿕ǃĐඈ %ǆͼȸ^ඈ Ě 4 Ȉඈ̓ ¾ඊྡྷඈ ༘Ȍඈ ᢪ௤ ϣ⑃ּ͟ᖏՁ 43 ȖüḰơՈᜬẂॅ 2 a a Figure 4 The gene transcription expression of synaptophysin and ϣ⑃ּ͟ᖏՁ 43 growth associated protein 43 in

Ẃ b b

ᜬ 1 b the rat brain

ü b Ěͼ^͢Ņ 3 ඈɨṇ aP < 0.05 GAPDH Ȗ b %ǆ ^࿕ǃĐඈɨṇ P < 0.054 ࿕ǃĐඈ ¾ඊྡྷඈ ༘Ȍඈ ͼȸ^ඈ 0 ᢪ௤ ϣ⑃ּ͟ᖏՁ 43ॅ

࿕ǃĐඈ %ǆͼȸ^ඈ 9࿕ඈඋॅᢪ௤ǎ۔Ě 5 Ȉඈ̓ ¾ඊྡྷඈ ༘Ȍඈ 3 ᢪ௤ ϣ⑃ּ͟ᖏՁ 43 ՈᖏՁᜬẂॅ Figure 5 Expression of a a synaptophysin and growth

2 ϣ⑃ּ͟ᖏՁ 43 Ẃ associated protein 43 proteins in ᜬ b b the rat brain Ձ b b

ᖏ 1 Ěͼ^͢Ņ 3 ඈɨṇ aP < 0.05 GAPDH ^࿕ǃĐඈɨṇ bP < 0.054 %ǆ ࿕ǃ Đඈ ¾ඊྡྷඈ ༘Ȍඈ 0 ͼȸ^ඈ ᢪ௤ ϣ⑃ּ͟ᖏՁ 43ॅ

ɪȆȔ ŵΛÛA˵ʩĉ"43Ά9Ǉǳĝ2ê༘ ඈ3%ǆͼȸ^ඈॅᢪ௤ǎϣ⑃ּ͟ᖏՁ43ᜬẂʌz࿕ǃ 2.6 Ȍඈॅᢪ௤ǎϣ⑃ּ͟ᖏՁ43ᜬẂºጛʌz࿕ǃĐඈ3% Đඈ(P < 0.05) ᢅĚ54 ǆͼȸ^ඈʐ¾ඊྡྷඈ(P < 0.05) ¾ඊྡྷඈ3%ǆͼȸ^ ඈॅᢪ௤ǎϣ⑃ּ͟ᖏՁ43ᜬẂʌz࿕ǃĐඈ(P < 0.05) 3ᩬ᩾Discussion ᢅĚ44 9࿕ǃĐĆš᰻ࠢඓඊྡྷ]ǳₑņɳՈǃĐ ᪵Ǖ࿕ 2.7 ɪȆȔ ŵΛÛA˵ʩĉ"43ĉ"ĝ2ê ༘Ȍඈ ඈඋՈʻĚàɬ਍ ẟ໐5྅˯້Ոϣʁƽ͔[28-30]4% ǳęûʻĚàɬÑ෗ᢧ9࿕ǃ ᢪ௤ǎϣ⑃ּ͟ᖏՁ43ʌz͢Â3ඈ(P < 0.05) ¾ඊྡྷ ǆȯƅ̶࢑ûʻĚtॅ

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Ոࠢඓ£࿁ Ǹǳ࿁۔ΝΈrₑƧ9࿕ǃĐµƧ̓ ۔̓ Đ[31-34]Ǹ̲͢ȭࠢඓ¾ඊྡྷՈʺȪϏĚ ỞẋƯẟࠢඓ ࿕ǃĐľ࿕ඈඋॅᢪ௤ǎϣ⑃ּ͟ᖏՁ43۔ඊྡྷՈňϬÑǚ̩ǃĐՈࠢඓඊྡྷ4ࠢඓ¾ඊྡྷ«P࢑ ỞẋƯẟ̓¾ ǳÑȕࠢඓ̯ ĚՈ¾ඊྡྷ ᪩ඊྡྷẜǳÑẟᜐႮuŰ, ՈᜬẂˈ¿ǕƉňϬ4 ʐႮuමť Â ̶͠࢑ࠢඓüƄÑΝΈ9࿕ǃĐ[35-37]4 Ĳϵzࠢඓ¾ඊྡྷńࢿΉẋ࣏ƌϏƅ└ ͢ࢿΉՈ͏ћ ň້ᯥǲǎ̓Ɏ̯Y̓ôκÉαôèͯʑΛfɻ -֎ΞॅƖuȔŀʃȅRæɻģʕ˵ʩŶ-Ȕ άȘKǛ4rṇ̓└:4໐%ǆͼȸ^ǳỞẋʺȪࠢඓ Ēˊ⒲LôκˎiȎ;Ƥˋεĝ͸%Θ̠ͮʩŦʦȎŨ ඊྡྷƯẟȒ້ࢿΉՈћά4ǸƵۘऺՈֲՈńᢆȓx້ ȖŶĝȔŀŷ˂ʃõ˼ıΛˋνƽʷôκŦʦÝȠȊ˗Y̓¾ Ȍȭ9࿕ǃĐՈƽˑ4 ƖuĝĕĴKE`ϝ˽ſȋî˳ſıϠºΓǖȍȍý˼ŝɏɻּ༘ ǸƵۘऺº߾ %ǆͼȸ^^ࠢඓ¾ඊྡྷࢿΉּ༘Ȍ ģĝǈ̧%Θ͑ȷ̢ɻģȎėƤŦʦôκǠˍôκΡ~Qəʷǋ ŷ˂(ARRIVE ŷ˂) ໐řư͏ћඈȭࠢ ࠢඓ£࿁෾ǃ᪈۔ºጛ┑ĺr̓ তɡₑɻģ1νlˎ CNKI ǉɘϡɻˬϗ!ˊȌY̓ 3 ඓǃĐՈ£࿁ʮ̩KĄz9࿕ǃĐඈ ẝȴ߾༘Ȍ͏ћȴ É(Ɨ Ոࠢඓ£࿁4໐Morrisˈẻǟǒɀ඗Ș ত̲Ǖɻģlˋˍ̔ȅ̓ȻÔǅνňȹȻÔKȐ˾ÌƷɘ۔ʌr9࿕ǃĐ̓ ᜬ ༘Ȍඈ35 dr¿ţȈûrⒸṇ%ǆͼȸ^ඈǎ¾ඊ ǷŻ ň້̌ĳȔŀǎ̓ƋΡ~˼ǈ̧ĝȒɻƏ˫ĝ^Ƃ̓ɡ ᱎ¿ǴƵϔǎńֲʃ müóQȒɻƏÇΛĝ°ûȕ͛ļ(Gģαƀ·ļÿ)βǳΛृ ڱṇ࿕ǃĐඈº෭ ڱඈ෭ྡྷ ᬥ└ä·Სࡿ^ʇᲡࡿՂ ɨţʌz%ǆͼȸ^ඈǎ¾ඊ ϊcĶuʩʷşLáǫ̂˼̏ͦKæ4ě˺( ྡྷඈ ºʌz࿕ǃĐඈ ẝȴ߾%ǆͼȸ^ʐࠢඓ¾ඊ তČƿɻģ1νÖŽ.ǠȔŀę+NζįƤ1+˵ʩ ̠0 ՈᩴȒ࿁ʐᜐ&࿁۔ࢿΉ༘ȌǳƅάƯẟ9࿕ǃĐ̓ྡྷ Ō΢ቻǚ̌ŃĆ͙Çǚ;$ɻģKɻģ1νÖŽ. 4໐ᆓƤஂö൦ɏᄶ඗Șº߾%ǆͼȸ^ඈǎ¾ඊྡྷඈ ǠȔŀę+NζįƤ1+˵ʩ̠0ɬũõʃ̂͠æ̠0 į ࿕ǃĐľඈඋाβṇɃ ༘Ȍඈ࿕ǃĐľඈඋाβό:4 ʐÃǜϤ͇øhÃ˵ȅǒüʃ̂3.0ǰċKÝ˾ŨDhĝē˗K ͍ ࿕ǃĐඈǳᢅ࿕ǃĐľඈඋाβº ẝᜬ༘Ȍ͏ Í͠ƘNǜϤ͇ʲĝΆ°ɻˍ`%ΘŐŜ˼ĤĚKȅðÍ͠ Q˽h̴ÇŸ˗ÛÚ]¦ĶíFϗƔIΡƍ4ȶɻˬK ћƯẟr9࿕ඈඋՈǚ̩ʐࠢඓඊྡྷՈ ĚʺȪ4ǒɀ Eɡŭ ŻƔDKhȔpĝģnļͿΓƙQ˽˾ƽhȗ ᢆȓ4%ǆͼȸ^ºጛƯẟr̲ļɳࠢඓ¾ඊྡྷࢿΉȒՈ ƌϏʐ O ẝȴ߾%ǆͼȸ^ǳỞẋȴʌࠢඓ¾ඊྡྷʺ 4 ǆ໇ǲ References ǆͼȸ^ºጛ [1] Diaz-Arrastia R,Kenney K.Epidemiology of traumatic brain% کȪʐƌϏȴʌ͢ћά4ǒǒɀ඗Șǳ ȴʌrࠢඓ¾ඊྡྷń࿕ǃĐľǻՈƌϏʐ O ẝ^ injury.Handb Clin Neurol.2015;127:3-13. [2] Haring RS,Narang K,Canner JK,et al.Traumatic brain injury in ¾ǲՈۘऺṇ&PႸ[26-27] %ǆͼȸ^ՈàϬᢧΟrࠢඓ the elderly:morbidity and mortality trends and risk factors.J ඊྡྷńࢿΉẋ࣏ƌϏɋĺՈ⒲L4ࠢඓ¾ඊྡྷỞẋǆ^ Surg Res.2015;195(1):1-9. .Ոᜐ&ƚ࿁4ẝP [3] Fernandezrodriguez E,Bernabeu I,Castro AI,et al۔ࠢඓǃĐǚ̩ºጛΝΈr࿕ǃĐ̓ ƶ:ǳ࿁^ࠢඓ¾ඊྡྷȭࠢඓ᫇ᅆüƄՈᜬẂՈƯẟňϬ Hypopituitarism after traumatic brain injury.Endocrinol Metab Clin North Am.2015;44(1):151-159. ƅ͟4ۘऺᜬ ࠢඓ¾ඊྡྷǳºጛȴʌϣ⑃ּ͟ᖏՁ43 ,Ạȃ.9࿕ǃĐ͏ћẟʉ[J].ȖɶĿƚ᩾ŷ,2014ٸ,ů,˅%ṍٸ [4] ʐॅᢪ௤Ո ͠4ẝ^ǒɀ༘ȌඈᢆȓՈǜĚṇ&PႸ4 18(1):107-109. :Ȍඈϣ⑃ּ͟ᖏՁ43ʐॅᢪ௤ՈᜬẂˈ¿ºṇʌ4 [5] Yoshida Y,Yamanaka S.Recent stem cell advances༘ ϣ⑃ּ͟ᖏՁ43«Ëͯƌńzࠢඓඊྡྷࠢඓ᫇ᅆüƄ induced pluripotent stem cells for disease modeling and stem cell–based regeneration. Circulation. 2010;122(1): ͢ǳƯẟ࿕ǃĐȒՈࠢඓிඣՈǚ̩ Âᝯᩨ&«ࠢඓ͹ 80-87. [38] :(ċ.ࠢඓ¾ඊྡྷۘऺẟʉǎQë[J].࢕äǍʻ,2011,28(16 ϣՈʃȣ 4᪩඗Șȴ߾༘Ȍ͏ћ෗ᢧr࿕ˈྃ ÂƯẟ [6] rࠢඓඊྡྷՈϣ⑃ʐǚ̩ ȭ࿕̓ȏ᰻4r͏ћňϬ4ॅ 10172-10172. [7] Gage FH,Temple S.Neural stem cells:generating and ᢪ௤«ॅᢪɃ͵Ո£࿁ɳᖏՁ ͢ǳň&ࠢඓிඣॅ regenerating the brain.Neuron.2013;80(3):588-601. ,Ƅʃᩴʐ£࿁ɳʃȣ ͢ᜬẂˈ¿ǳĿɴϦॅᢪՈ [8] ᰂ͔,⒯¡Œ,ȸ)Y.ࠢඓ¾ඊྡྷۘऺẟʉ[J].ĿƚවẴ ᢪՈ ϔₓʐ£࿁[39-46]4ẝᜬ༘Ȍ͏ћńₑņࠢඓඊྡྷʐǚ̩ 2010,16(8):1149-1151. ඓẋ%ǆͼȸ^ՈƯ [9] Kim SU.Neural stem cell-based gene therapy for brain کࠢඓ£࿁5☦ћάºጛ4ǒǸǳ tumors.Stem Cell Rev Rep.2011;7(1):130-140. ẟňϬ ࠢඓ¾ඊྡྷՈʺȪϏĚ࿁ǣ4rȴʌ ͢ΝΈ ,5Ò,┌,ϣ.ࠢඓ¾ඊྡྷּۘ͟ऺẟʉ[J].ęĿሳūś [10] .͠ʐᜬẂ ȴʌȒՈϣ⑃ּ͟ᖏՁ43 2011,9(8):44-46 rࠢඓ᫇ᅆüƄՈ ՈࠢඓǃĐ ƯẟrࠢඓඊྡྷՈǚ̩ [11] Xie C,Cong D,Wang X,et al.The effect of simvastatin۔ʐॅᢪ௤෗ᢧr̓ ʐϣ⑃ ºʺ¤rࠢඓॅᢪՈϔₓ ȴʌrࠢඓॅᢪՈ treatment on proliferation and differentiation of neural stem cells after traumatic brain injury.Brain Res.2014;1602:1-8. £࿁4ẟ໐ %ǆͼȸ^^ࠢඓ¾ඊྡྷ༘Ȍ͏ћºጛΝΈ [12] Ahmed A,Shtaya A,Zaben M,et al.Activation of endogenous ࿕ඈඋǃĐ4 neural stem cells after traumatic brain injury.Lancet.2014۔ᜐ&ƚʐᩴȒ࿁ ෗ᢧr̓۔r̓ ǒɀỞẋ%ǆͼȸ^^ࠢඓ¾ඊྡྷࢿΉ͏ћ9࿕ǃĐ 383(383):S18.

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