實(shí)體瘤不僅由惡性細(xì)胞組成，而且是復(fù)雜的器官樣結(jié)構(gòu)，包含多種細(xì)胞類型，包括多種遷移性造血細(xì)胞和常駐基質(zhì)細(xì)胞。白細(xì)胞浸潤到實(shí)體瘤中的作用在十多年前就被注意到了。這些細(xì)胞類型遷移到腫瘤中被解釋為宿主對(duì)生長中的腫瘤產(chǎn)生免疫反應(yīng)的證據(jù)，但最近很明顯，腫瘤在很大程度上被認(rèn)為是自身并且缺乏強(qiáng)抗原。相反，它們似乎被選中來操縱宿主免疫系統(tǒng)以防止排斥反應(yīng)并利用它來促進(jìn)自己的生長和傳播。這導(dǎo)致提出由骨髓細(xì)胞、中性粒細(xì)胞、樹突狀細(xì)胞 （DC）、嗜酸性粒細(xì)胞、肥大細(xì)胞、淋巴細(xì)胞和巨噬細(xì)胞組成的造血細(xì)胞浸潤在致癌作用中起因果作用。從廣泛的實(shí)體瘤中收集的臨床數(shù)據(jù)強(qiáng)調(diào)了這些發(fā)現(xiàn)，因?yàn)楦呙芏鹊陌准?xì)胞浸潤，尤其是巨噬細(xì)胞，與疾病的不良預(yù)后相關(guān)。

腫瘤相關(guān)巨噬細(xì)胞 （TAM） 產(chǎn)生大量促進(jìn)腫瘤發(fā)生的因子。突出的包括堿性成纖維細(xì)胞生長因子 （bFGF）、血管內(nèi)皮生長因子 （VEGF）、血小板衍生生長因子 （PDGF）、轉(zhuǎn)化生長因子 β （TGFβ）、血管生成素（Ang1 和 Ang2）、IL-1 和 IL-8 等白細(xì)胞介素、腫瘤壞死因子-α （TNF-α）、胸苷磷酸化酶 （TP）、基質(zhì)金屬蛋白酶 MMP-9 和 MMP-2、一氧化氮 （NO） 和趨化因子 。這些分子的協(xié)調(diào)空間和時(shí)間表達(dá)導(dǎo)致內(nèi)皮細(xì)胞 （EC） 的增殖和遷移、細(xì)胞外基質(zhì)的重塑和穩(wěn)定血管的形成。巨噬細(xì)胞容易促進(jìn)這些過程，因?yàn)樗鼈兊膯魏思?xì)胞前體遷移到特定位置，例如缺氧腫瘤組織，在那里他們分化和合成血管生成分子。腫瘤需要血管生成才能長出超過幾毫米的大小。血管生成還被發(fā)現(xiàn)通過提供氧氣和營養(yǎng)以及清除廢物對(duì)廣泛的腫瘤生長和轉(zhuǎn)移至關(guān)重要。VEGF 生長因子家族由結(jié)構(gòu)高度相關(guān)的蛋白質(zhì)及其相應(yīng)的受體組成，在血液系統(tǒng)惡性腫瘤和實(shí)體瘤的血管生成過程中起著至關(guān)重要的作用。幾種靶向 VEGF 或其受體的治療方法顯示出良好的臨床結(jié)果。此外，最近的研究表明，由募集骨髓衍生的血管白細(xì)胞介導(dǎo)的血管發(fā)生，這些血管白細(xì)胞同時(shí)表達(dá)內(nèi)皮細(xì)胞和樹突狀細(xì)胞標(biāo)志物并分化為內(nèi)皮樣細(xì)胞，在腫瘤血管生成中起著重要作用。Maruyama 等人新近發(fā)表的一篇論文記錄了巨噬細(xì)胞在病理性淋巴管生成中起的關(guān)鍵作用，他們提供了證據(jù)，證明 CD11b+ 巨噬細(xì)胞能夠轉(zhuǎn)分化為淋巴內(nèi)皮細(xì)胞簇，這些細(xì)胞簇在小鼠角膜移植模型中加入現(xiàn)有的淋巴管。

雙膦酸鹽是臨床上用于預(yù)防或抑制骨轉(zhuǎn)移或骨過度吸收發(fā)展以及治療類風(fēng)濕性關(guān)節(jié)炎和骨關(guān)節(jié)炎等炎癥性疾病的化合物。最近，已發(fā)現(xiàn)使用雙膦酸鹽作為抗血管生成劑可以抑制實(shí)體瘤的生長和轉(zhuǎn)移（Giraudo 等人，2004 年）。隨著雙膦酸鹽氯膦酸鹽包埋到脂質(zhì)體 （Clodronate Liposoems，Nico Van Rooijen等人） 中，已經(jīng)開發(fā)出一種用于選擇性耗竭巨噬細(xì)胞的有效試劑，并成功應(yīng)用于多項(xiàng)免疫學(xué)研究。因此，我們研究了 TAMs 耗竭是否會(huì)抑制腫瘤血管生成，從而抑制腫瘤生長和播散的可能性。在這里，我們表明氯膦酸鹽脂質(zhì)體介導(dǎo)的 TAM 耗竭抑制腫瘤生長，可能是通過阻斷腫瘤血管生成。

體外實(shí)驗(yàn)

游離氯膦酸鹽：clodronate

脂質(zhì)體包裹的氯膦酸：liposome encapsulated clodronate (clodrolip)，Clodronate Liposomes

in vitro effect of free and liposome encapsulated clodronate (clodrolip). (A) Concentration-dependent cytotoxicity of clodrolip on macrophages (isolated from Sv129 mice by peritoneal lavage) in vitro. (B) Cytotoxicity of clodronate or clodrolip on different cells in vitro. Macrophages, HUVE, F9 and A673 cells were cultured in the presence of 1?mg?ml?1 clodronate or clodrolip for 6?h. Results are means±s.e.m. (n=3). Statistical analysis: *P<0.05 vs untreated cells.

體內(nèi)實(shí)驗(yàn)

in vivo effect of free and liposome encapsulated clodronate (clodrolip)。(C) Selective depletion of spleen cell populations after treatment with clodronate and clodrolip. Spleen tissues obtained from immunocompetent Sv129 mice injected with PB, clodronate or with clodrolip are shown (initial dose 2?mg?20?g?1 mouse body weight, followed by 1?mg, every 4 days, i.p.). Spleens were removed and sections IHC stained for marginal zone metallophilic MOMA1+, marginal zone ER-TR 9+, red pulp F4/80+, CD68+ and CD11b+ macrophages, the DC subsets FDC+ and CD11c+, B220+ B cells, and CD3+ T cells. Bar: 100?μm.

顯而易見，Cloddrolip組清除巨噬細(xì)胞效果更優(yōu)，如上圖所示，F(xiàn)4/80陽性的巨噬細(xì)胞相比較對(duì)照組PBS和游離氯膦酸鹽組，簡直碾壓。同時(shí)也證明了游離氯膦酸鹽無法體內(nèi)清除巨噬細(xì)胞。因此，建議訂購荷蘭Liposoma商業(yè)化巨噬細(xì)胞清除劑--Clodronate Liposomes,氯膦酸鹽脂質(zhì)體。貨號(hào)CP-005-005. 同時(shí)，相比較對(duì)照組PBS，氯膦酸鹽脂質(zhì)體對(duì)其他免疫細(xì)胞沒有清除效果?？梢钥次覀冎暗奈恼?，巨噬細(xì)胞清除劑會(huì)清除外周血其他細(xì)胞嗎？巨噬細(xì)胞清除劑會(huì)清除骨髓其他細(xì)胞嗎？

Clodrolip（Clodronate Liposomes） promotes macrophage depletion and inhibits tumour growth

We next studied the effects of clodrolip on tumour progression and angiogenesis in the highly vascularised and fast-growing syngeneic F9 teratocarcinoma mouse tumour model. Mice were treated i.p. with PB, empty liposomes, clodronate or clodrolip. The initial clodronate dose was 2?mg?20?g?1 mouse body weight, followed by 1?mg?20?g?1 mouse body weight given every 4 days. Therapy onset was 6?h after tumour cell inoculation. In two groups, clodrolip therapy was delayed to days 4 and 8, respectively. Clodrolip treatment inhibited tumour growth, even at the most delayed therapy onset (days 8 and 12) (Figure 2A and Supplementary Figure s2A). The most effective growth inhibition (74%, P=0.0185) was obtained with an early treatment on days 0, 4, 8 and 12. Clodronate (5?mg) given on days 0, 4, 8, 12 had an insignificant inhibitory effect of 45% (P=0.21), comparable to the days 4, 8 and 12 delayed clodrolip schedule (49%, P=0.167). The delayed onset of therapy at time points where tumours were already established and well vascularised (day 4 or 8) resulted in less pronounced growth inhibition, suggesting that macrophage depletion in large tumours is not sufficient to efficiently inhibit tumour growth.

荷蘭Nico Van Rooijen教授First開發(fā)了該試劑，也就是現(xiàn)在的荷蘭Liposoma品牌，國內(nèi)客戶可以直接訂購巨噬細(xì)胞清除劑：氯膦酸鹽脂質(zhì)體Clodronate Liposomes氯膦酸二鈉脂質(zhì)體，訂購貨號(hào)CP-005-005。規(guī)格是5ml清除劑+5ml對(duì)照試劑。國內(nèi)客戶可以聯(lián)系大中華辦事處靶點(diǎn)科技（Target Technology）。專業(yè)的技術(shù)團(tuán)隊(duì)給您的巨噬細(xì)胞清除實(shí)驗(yàn)以解決方案，以及根據(jù)結(jié)果給與優(yōu)化和建議。