3. “多巴胺D2受體”研究為治療“第一晚效應”提供新思路
【摘要】
醫(yī)學上把一些人換床后無法入睡的現(xiàn)象稱為“第一晚效應”。資料顯示，隨著工業(yè)化進程的加快，社會競爭、工作壓力、不良夜生活習慣及人口老齡化等原因，全球三分之一的人存在睡眠問題，其中不少人因經(jīng)常出差不能在習慣的床上睡眠或入睡前情緒改變、精神亢奮或緊張而難以入眠，深陷“第一晚效應”的痛苦之中不能自拔，嚴重影響到次日工作效率和身體健康。復旦大學醫(yī)學神經(jīng)生物學國家重點實驗室黃志力課題組研究人員曲衛(wèi)敏副教授、徐昕紅博士等運用高度自動化睡眠覺醒解析系統(tǒng)，記錄已經(jīng)基因剔除“多巴胺D2受體”小鼠的睡眠過程，并結合藥理學等手段，從基因到行為解析了多巴胺D2受體在睡眠覺醒調控中的作用。結果發(fā)現(xiàn)，與正常小鼠（野生型小鼠）相比，剔除了多巴胺D2受體的小鼠，活動期維持覺醒困難，睡眠量增加。 為模擬人在新環(huán)境下如出差住宿等，出現(xiàn)換床后失眠現(xiàn)象，即“第一晚效應”，研究人員更換動物居住環(huán)境，考察小鼠在新環(huán)境中的睡眠行為。結果顯示，多巴胺D2受體正常的小鼠面對新環(huán)境刺激，極為不習慣、入睡困難，而剔除了多巴胺D2受體的小鼠則“高枕無憂”，迅速入睡。

【點評】
多巴胺D2受體刪除的老鼠實驗中不出現(xiàn)”第一晚效應”, 在新環(huán)境中能迅速入睡，且睡眠量增加。 這個現(xiàn)象是否與生物鐘有關，能否在改善睡眠上有利用價值，值得研究一下，看是否可能用在生物鐘飲食療法上。不過需要注意的是，通過基因敲除技術研究的結論只是單個（或某幾個）基因的作用，無法考慮或排除整體的調控在其中的影響，因此結論往往不是那么十分確定的。

【原文摘錄】
The Journal of Neuroscience, March 24, 2010, 30(12):4382-4389; doi:10.1523
Essential Role of Dopamine D2 Receptor in the Maintenance of Wakefulness, But Not in Homeostatic Regulation of Sleep, in Mice
Wei-Min Qu,1 Xin-Hong Xu,1 Ming-Ming Yan,1 Yi-Qun Wang,1 Yoshihiro Urade,2 and Zhi-Li Huang1
Dopamine (DA) and its D2 receptor (R) are involved in cognition, reward processing, and drug addiction. However, their roles in sleep–wake regulation remain unclear. Herein we investigated the role of D2R in sleep–wake regulation by using D2R knock-out (KO) mice and pharmacological manipulation. Compared with WT mice, D2R KO mice exhibited a significant decrease in wakefulness, with a concomitant increase in non-rapid eye movement (non-REM, NREM) and REM sleep and a drastic decrease in the low-frequency (0.75–2 Hz) electroencephalogram delta power of NREM sleep, especially during the first 4 h after lights off. The KO mice had decreased mean episode duration and increased episode numbers of wake and NREM sleep, many stage transitions between wakefulness and NREM sleep during the dark period, suggesting the instability of the wake stage in these D2R KO mice. When the KO mice were subjected to a cage change or an intraperitoneal saline injection, the latency to sleep in the KO mice decreased to half of the level for WT mice. The D2R antagonist raclopride mimicked these effects in WT mice. When GBR12909, a dopamine transport inhibitor, was administered intraperitoneally, it induced wakefulness in WT mice in a dose-dependent manner, but its arousal effect was attenuated to one-third in the D2R KO mice. However, these 2 genotypes showed an identical response in terms of sleep rebound after 2, 4, and 6 h of sleep deprivation. These results indicate that D2R plays an essential role in the maintenance of wakefulness, but not in homeostatic regulation of NREM sleep.

4. 番茄基因加抗艾滋藥物的新基因療法治療癌癥
【摘要】
　　瑞典研究人員最新發(fā)現(xiàn)，一種番茄基因與藥物組合后能破壞癌細胞，這一發(fā)現(xiàn)將有助于用基因療法治療癌癥。瑞典隆德大學研究人員日前發(fā)表公報說，這種番茄基因在幫助建立和修復番茄基因組方面“非?；钴S”，但它本身并不足以破壞癌細胞。在先后測試了不同藥物后，研究人員最終發(fā)現(xiàn)，這種番茄基因與抗艾滋病藥物AZT組合后，能更有效地打擊癌細胞。研究人員指出，很多人對基因療法心存疑慮，擔心病人的基因在接受治療后發(fā)生改變，引發(fā)更多的不良反應。然而，上述研究并不存在這種風險 ，因為番茄基因僅僅被注入癌細胞內，并不影響其他細胞。 新華網(wǎng) 2010-3-29 12:11:30

【點評】
　　細胞培養(yǎng)以及裸鼠實驗顯示 西紅柿TK1基因與抗艾滋藥物逆轉錄酶抑制劑AZT組合的自殺基因療法結合干細胞介導的基因注入 顯著提高了癌細胞對藥物的敏感度，實質性的抑制了腫瘤生長，可以說在針對癌細胞的攻擊方面這的確是個很好的策略，只是一方面還僅僅在實驗動物身上看到效果，另一方面，它并未有顯示出可以治愈癌癥的潛力，而且將異種基因轉入動物體內哪怕只是體內的癌細胞中會產生什么后果尚未可知，也就存在著未知的風險。最基本的，這種策略依然是治標不治本。

【原文摘錄】
Neuro Oncol. 2010 Feb 13 PMID: 20154339
Plant thymidine kinase 1: a novel efficient suicide gene for malignant glioma therapy.
Khan Z, Knecht W, Willer M, Rozpedowska E, Kristoffersen P, Clausen AR, Munch-Petersen B, Almqvist PM, Gojkovic Z, Piskur J, Ekstr?m TJ.
The prognosis for malignant gliomas remains poor, and new treatments are urgently needed. Targeted suicide gene therapy exploits the enzymatic conversion of a prodrug, such as a nucleoside analog, into a cytotoxic compound. Although this therapeutic strategy has been considered a promising regimen for central nervous system (CNS) tumors, several obstacles have been encountered such as inefficient gene transfer to the tumor cells, limited prodrug penetration into the CNS, and inefficient enzymatic activity of the suicide gene. We report here the cloning and successful application of a novel thymidine kinase 1 (TK1) from the tomato plant, with favorable characteristics in vitro and in vivo. This enzyme (toTK1) is highly specific for the nucleoside analog prodrug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. An important feature of toTK1 is that it efficiently phosphorylates its substrate AZT not only to AZT monophosphate, but also to AZT diphosphate, with excellent kinetics. The efficiency of the toTK1/AZT system was confirmed when toTK1-transduced human glioblastoma (GBM) cells displayed a 500-fold increased sensitivity to AZT compared with wild-type cells. In addition, when neural progenitor cells were used as delivery vectors for toTK1 in intracranial GBM xenografts in nude rats, substantial attenuation of tumor growth was achieved in animals exposed to AZT, and survival of the animals was significantly improved compared with controls. The novel toTK1/AZT suicide gene therapy system in combination with stem cell-mediated gene delivery promises new treatment of malignant gliomas.

5. 一種可使藥物直接攻擊癌細胞的全新方法
【摘要】
　　加拿大蒙特利爾大學和拉瓦爾大學的科學家發(fā)現(xiàn)了一種可使藥物直接攻擊癌細胞的全新方法，其可為急性骨髓白血病患者等癌癥病人帶來福音。據(jù)科學家稱，這種新方法目前已接近于臨床試驗。相關文章發(fā)表在最新出版的《生物化學雜志》上。 研究負責人、蒙特利爾大學藥學系教授丁戴爾•拉門塔爾表示，他們發(fā)現(xiàn)人體中部分類型的細胞存在一個“門口”，如源自骨髓的細胞就存在一個允許博來霉素等抗癌藥物進入的“門”，找到并打開這扇“門”就可讓藥物直接攻擊引發(fā)白血病的癌細胞。該成果為癌癥治療開辟了一條新途徑。
　　拉門塔爾教授介紹，他在十年前開始將該理論付諸實踐，在與人體細胞十分接近的發(fā)酵用酵母上進行了試驗。目前所獲發(fā)現(xiàn)正是基于酵母實驗的成果，新方法可被應用于人體細胞，并能很快進入臨床治療。
　　據(jù)介紹，新方法對于癌癥患者特別是急性骨髓白血病患者實屬福音。急性骨髓白血病影響人的白細胞，這種癌癥非常難治療，絕大部分患者對各種抗癌藥物沒有反應。拉門塔爾教授表示，新方法可以將抗癌藥劑以束流的形式治療急性骨髓白血病。他說：“例如我們發(fā)現(xiàn)博來霉素等抗癌藥劑對來自患者身上的淋巴瘤細胞具有正面結果，但同時還要依靠‘門口’的存在。”由于博來霉素不表現(xiàn)為免疫抑制劑，他認為這對患者來說是一個十分利好的消息。
拉門塔爾教授還提醒到，新找到的“門口”只存在于部分細胞類型，比如那些來自于骨髓的細胞，但對于乳腺癌等就不起作用，這樣就很難使用博來霉素等來治療乳腺癌患者。因此，他認為目前應著手尋找能夠刺激“門口”產生的方式，這樣才能夠使用博來霉素等藥物治療更多類型的癌癥

【點評】
　　尋找能夠刺激“門口”產生的方式是沒準的事情，要尋找只刺激癌細胞“門口”產生的方式，不影響正常細胞，更難?？傊荒芙鉀Q藥物對癌細胞和正常細胞的相似作用，化療的前景就無法看好。如果能像再生營養(yǎng)物質那樣在有利于正常細胞的同時消滅癌細胞，把看似矛盾的兩個方面統(tǒng)一起來做到一舉兩得。這才是癌癥治療的最好結果。

【原文摘錄】
JBC doi: 10.1074/jbc.M109.046151
The Human Carnitine Transporter SLC22A16 Mediates High Affinity Uptake of the Anticancer Polyamine Analogue Bleomycin-A5*
Mustapha Aouida,1, Richard Poulin§ and Dindial Ramotar,2
Bleomycin is used in combination with other antineoplastic agents to effectively treat lymphomas, testicular carcinomas, and squamous cell carcinomas of the cervix, head, and neck. However, resistance to bleomycin remains a persistent limitation in exploiting the full therapeutic benefit of the drug with other types of cancers. Previously, we documented that the Saccharomyces cerevisiae L-carnitine transporter Agp2 is responsible for the high affinity uptake of polyamines and of the polyamine analogue bleomycin-A5. Herein, we document that the human L-carnitine transporter hCT2 encoded by the SLC22A16 gene is involved in bleomycin-A5 uptake, as well as polyamines. We show that NT2/D1 human testicular cancer cells, which highly express hCT2, are extremely sensitive to bleomycin-A5, whereas HCT116 human colon carcinoma cells devoid of detectable hCT2 expression or MCF-7 human breast cancer cells that only weakly express the permease showed striking resistance to the drug. NT2/D1 cells accumulated fluorescein-labeled bleomycin-A5 to substantially higher levels than HCT116 cells. Moreover, L-carnitine protected NT2/D1 cells from the lethal effects of bleomycin-A5 by preventing its influx, and siRNA targeted to hCT2 induced resistance to bleomycin-A5-dependent genotoxicity. Furthermore, hCT2 overexpression induced by transient transfection of a functional hCT2-GFP fusion protein sensitized HCT116 cells to bleomycin-A5. Collectively, our data strongly suggest that hCT2 can mediate bleomycin-A5 and polyamine uptake, and that the rate of bleomycin-A5 accumulation may account for the differential response to the drug in patients.

6. C60運載基因技術為糖尿病患者送福音
【摘要】
　　日本東京大學的研究人員首次開發(fā)出了利用超小球形碳分子C60（富勒烯）導入基因的新技術。該技術有望為糖尿病患者帶來福音。C60是60個碳原子結合在一起形成的直徑不足1納米的球狀微小粒子。東京大學副教授野入英世和教授中村榮一率領的研究小組讓C60攜帶4個氨基，制造出了水溶性C60，使其與基因結合成為可能。
　　研究人員將結合了綠色熒光蛋白基因的水溶性C60注射到實驗鼠體內。結果發(fā)現(xiàn)實驗鼠的肺、肝和脾都出現(xiàn)了該基因，證實了水溶性C60具有強大的基因運載能力 。在隨后的實驗中，研究人員讓水溶性C60攜帶指導合成胰島素的基因進入實驗鼠體內，結果實驗鼠體內的胰島素水平增加到平常的1.5倍，血糖值也降低了20%以上。研究人員介紹說，與基因結合的水溶性C60穿過細胞膜以后就會與基因分離，隨尿液排出體外，不會在體內堆積。
　　目前，治療糖尿病的手段主要是通過給患者直接注射胰島素來降低血糖值。日本研究人員認為，此次開發(fā)的新技術達到實用化水平后，降低血糖值效果的持續(xù)時間將比直接注射還要長，由此將大大減輕患者的負擔。另外，這項新技術還有可能促成安全性更高的基因治療糖尿病方法的出現(xiàn)。

【點評】
　　該技術若能發(fā)展成熟，可能會為RNAi技術更好的用于需基因治療的疾病提供很大幫助?？傊?，這看上去是一項很好的基因運載技術。

【原文摘錄】
PNAS doi: 10.1073/pnas.0909223107
In vivo gene delivery by cationic tetraamino fullerene
Rui Maeda-Mamiyaa,b, Eisei Noirib,1, Hiroyuki Isobec, Waka Nakanishic, Koji Okamotob, Kent Doib, Takeshi Sugayad, Tetsuro Izumie, Tatsuya Hommaa, and Eiichi Nakamuraa,1
Application of nanotechnology to medical biology has brought remarkable success. Water-soluble fullerenes are molecules with great potential for biological use because they can endow unique characteristics of amphipathic property and form a self-assembled structure by chemical modification. Effective gene delivery in vitro with tetra(piperazino)fullerene epoxide (TPFE) and its superiority to Lipofectin have been described in a previous report. For this study, we evaluated the efficacy of in vivo gene delivery by TPFE. Delivery of enhanced green fluorescent protein gene (EGFP) by TPFE on pregnant female ICR mice showed distinct organ selectivity compared with Lipofectin; moreover, higher gene expression by TPFE was found in liver and spleen, but not in the lung. No acute toxicity of TPFE was found for the liver and kidney, although Lipofectin significantly increased liver enzymes and blood urea nitrogen. In fetal tissues, neither TPFE nor Lipofectin induced EGFP gene expression. Delivery of insulin 2 gene to female C57/BL6 mice increased plasma insulin levels and reduced blood glucose concentrations, indicating the potential of TPFE-based gene delivery for clinical application. In conclusion, this study demonstrated effective gene delivery in vivo for the first time using a water-soluble fullerene.

7. 基因療法恢復患眼疾小鼠視力
【摘要】
　　據(jù)國外媒體報道，來自美國紐約州布法羅市、俄亥俄州克利夫蘭市和俄克拉何馬州的科學家使用基因療法，改善具有視網(wǎng)膜色素變性疾病的老鼠視力。這一研究結果表明，科學家在使盲人恢復視力的道路上取得了長足的進步。據(jù)悉，《美國實驗生物學學會聯(lián)合會雜志》2010年4月刊上發(fā)表的一篇研究報告中，科學家詳細闡述了利用合成的納米顆粒，改善具有視網(wǎng)膜色素變性疾病老鼠視力的過程。視網(wǎng)膜色素變性是視網(wǎng)膜光感受器細胞和色素上皮細胞變性，從而導致夜盲和進行性視野缺損的一組具有臨床亞型的基因遺傳性致盲眼病。
　　研究小組成員，俄克拉何馬州奧克拉荷馬大學健康科學中心細胞生物學系萊西博士和她的同事一起，研究了一組帶有視網(wǎng)膜緩慢變性基因的老鼠。萊西和她的同事對這些老鼠進行了三種不同類型的治療方法：一種方法是用包含Rds基因的納米顆粒來治療，一種方法是用正?；騺碇委?，還有一種方法是通過生理鹽水來治療。 實施三種不同類型的治療方法后，研究人員將實驗老鼠和其它具有視網(wǎng)膜色素變性或視網(wǎng)膜緩慢變性疾病老鼠進行比較，從而分析得出實驗老鼠視網(wǎng)膜的功能和結構。研究人員發(fā)現(xiàn)，接受納米顆?；虔煼ǖ睦鲜?，其視覺功能得到改善，具有明顯愈合的跡象，而且這種效果到實驗結束都還保持完好，而接受正?；蚝蜕睇}水治療的老鼠，其視力不斷下降。上述實驗結果表明，納米顆粒是耐受性良好，并且是安全無副作用的治療方法。
　　研究人員稱，他們希望此研究結果可幫助治愈那些和視網(wǎng)膜色素變性、遺傳性疾病和后天視網(wǎng)膜疾病等導致失明的疾病。 《美國實驗生物學學會聯(lián)合會雜志》雜志主編，杰拉爾• 德韋斯曼說：“使盲人恢復視力曾經(jīng)被稱為奇跡。隨著我們對進化、遺傳學和納米技術理解的加深，這種神奇的治療方法將變得非常普遍?！?/FONT>

【點評】
　　盲人復明是圣經(jīng)中的神跡，是醫(yī)學上的難題，上述基因療法還只是在老鼠實驗中顯示了效力，不過也能給盲人患者帶來一絲期望，是基因治療的進步之一，雖然基因療法在臨床應用上還很不成熟，也不確定到底能否成熟起來，畢竟這是在干預人體自身的遺傳信息，會造成多大的影響，什么樣的影響，我們并不清楚。

【原文摘錄】
Published as doi: 10.1096/fj.09-139147. (The FASEB Journal. 2010;24:1178-1191.)
Gene delivery to mitotic and postmitotic photoreceptors via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa
Xue Cai*, Shannon M. Conley*, Zack Nash*, Steven J. Fliesler , , , Mark J. Cooper|| and Muna I. Naash*,1
The purpose of the present study was to test the therapeutic efficiency and safety of compacted-DNA nanoparticle-mediated gene delivery into the subretinal space of a juvenile mouse model of retinitis pigmentosa. Nanoparticles containing the mouse opsin promoter and wild-type mouse Rds gene were injected subretinally into mice carrying a haploinsufficiency mutation in the retinal degeneration slow (rds+/–) gene at postnatal day (P)5 and 22. Control mice were either injected with saline, injected with uncompacted naked plasmid DNA carrying the Rds gene, or remained untreated. Rds mRNA levels peaked at postinjection day 2 to 7 (PI-2 to PI-7) for P5 injections, stabilized at levels 2-fold higher than in uninjected controls for both P5 and P22 injections, and remained elevated at the latest time point examined (PI-120). Rod function (measured by electroretinography) showed modest but statistically significant improvement compared with controls after both P5 and P22 injections. Cone function in nanoparticle-injected eyes reached wild-type levels for both ages of injections, indicating full prevention of cone degeneration. Ultrastructural examination at PI-120 revealed significant improvement in outer segment structures in P5 nanoparticle-injected eyes, while P22 injection had a modest structural improvement. There was no evidence of macrophage activation or induction of IL-6 or TNF- mRNA in P5 or P22 nanoparticle-dosed eyes at either PI-2 or PI-30. Thus, compacted-DNA nanoparticles can efficiently and safely drive gene expression in both mitotic and postmitotic photoreceptors and retard degeneration in this model. These findings, using a clinically relevant treatment paradigm, illustrate the potential for application of nanoparticle-based gene replacement therapy for treatment of human retinal degenerations.—Cai, X., Conley, S. M., Nash, Z., Fliesler, S. J., Cooper, M. J., Naash, M. I. Gene delivery to mitotic and postmitotic photoreceptors via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa.

8. DAF-16基因同壽命密切相關

【摘要】
　　從遺傳學角度研究衰老機制的一組英國科學家4月1日在《公共科學圖書館•綜合》網(wǎng)站撰文指出，他們針對實驗室蠕蟲進行的研究表明，DAF-16基因同壽命、免疫力密切相關。由于很多動物和人體內都擁有DAF-16基因，該發(fā)現(xiàn)有助于更好地了解影響人類衰老和免疫功能的原因。全球各地的人們正在大踏步邁向衰老，給健康和社會保障體系提出了巨大挑戰(zhàn)。丹麥科學家去年進行的一項研究發(fā)現(xiàn)，富裕國家出生的嬰兒中，有一半將可以慶祝其百歲誕辰?？茖W家急切渴望能夠找到令人衰老的原因，據(jù)此研發(fā)出藥物幫助人們盡可能長壽，并在有生之年保持健康。英國伯明翰大學的羅賓•梅爾領導了這項研究。梅爾團隊比較了4種關系密切的蠕蟲的壽命、抗藥性以及免疫力情況，他們發(fā)現(xiàn)，這4種蠕蟲體內的DAF-16基因的活性存在巨大的差異。更重要的是，DAF-16活性的差異同壽命、抵抗力和免疫力相輔相成：DAF-16的活性越高，蠕蟲的壽命越長，抗感染的免疫力越好。梅爾表示，這表明，免疫力和衰老密切相關。物種之間的DAF-16基因的活性的差異對衰老和健康具有非常重要的影響，這或許可以解釋人與人之間的壽命為何不同。
　　梅爾稱，DAF-16在體內大多數(shù)細胞中都很活躍，它們同人體內的FoxO家族調節(jié)基因非常類似，科學家認為FOXO家族在動物細胞的分化、生長、增殖、代謝、免疫及衰老調節(jié)方面具有多樣性功能。英國生物技術和生物科學研究協(xié)會負責人道格拉斯•凱爾表示，這個發(fā)現(xiàn)將幫助科學家理解決定人類衰老的相關機制。
【點評】
　　在分子機制上研究衰老和免疫，期望能夠找到令人衰老的原因，據(jù)此研發(fā)出藥物幫助人們盡可能長壽，并在有生之年保持健康。在復雜的分子調控網(wǎng)絡層次是做這項研究，即使有希望，也還有很漫長的路要走。而在細胞水平上的研究，人體再生復原科學則已經(jīng)找到了預防衰老的途徑并正在用于人體養(yǎng)生。

【原文摘錄】
Phenotypic Covariance of Longevity, Immunity and Stress Resistance in the Caenorhabditis Nematodes
Francis R. G. Amrit, Claudia M. L. Boehnisch, Robin C. May*
Abstract
Background
Ageing, immunity and stresstolerance are inherent characteristics of all organisms. In animals, these traits are regulated, at least in part, by forkhead transcription factors in response to upstream signals from the Insulin/Insulin–like growth factor signalling (IIS) pathway. In the nematode Caenorhabditis elegans, these phenotypes are molecularly linked such that activation of the forkhead transcription factor DAF-16 both extends lifespan and simultaneously increases immunity and stress resistance. It is known that lifespan varies significantly among the Caenorhabditis species but, although DAF-16 signalling is highly conserved, it is unclear whether this phenotypic linkage occurs in other species. Here we investigate this phenotypic covariance by comparing longevity, stress resistance and immunity in four Caenorhabditis species.
Methodology/Principal Findings
We show using phenotypic analysis of DAF-16 influenced phenotypes that among four closely related Caenorhabditis nematodes, the gonochoristic species (Caenorhabditis remanei and Caenorhabditis brenneri) have diverged significantly with a longer lifespan, improved stress resistance and higher immunity than the hermaphroditic species (C. elegans and Caenorhabditis briggsae). Interestingly, we also observe significant differences in expression levels between the daf-16 homologues in these species using Real-Time PCR, which positively correlate with the observed phenotypes. Finally, we provide additional evidence in support of a role for DAF-16 in regulating phenotypic coupling by using a combination of wildtype isolates, constitutively active daf-16 mutants and bioinformatic analysis.
Conclusions
The gonochoristic species display a significantly longer lifespan (p<0.0001) and more robust immune and stress response (p<0.0001, thermal stress; p<0.01, heavy metal stress; p<0.0001, pathogenic stress) than the hermaphroditic species. Our data suggests that divergence in DAF-16 mediated phenotypes may underlie many of the differences observed between these four species of Caenorhabditis nematodes. These findings are further supported by the correlative higher daf-16 expression levels among the gonochoristic species and significantly higher lifespan, immunity and stress tolerance in the constitutively active daf-16 hermaphroditic mutants。