小編說在前面的話：Bone remodeling is performed by osteoclasts and osteoblasts at the bone surface. Inside of bone is a network of numerous osteocytes, whose specific function has remained an enigma.
小編看到「enigma」這個單詞想到了一個很有趣的事情，相關故事附在了文末作為驚喜（請務必先閱讀文獻，勿直接跳到文末尋找答案，不然小心「驚喜」變「驚嚇」哦！）。
發表情況：
Cell Metabolism.2007 Jun; 5(6):464-75
Targeted ablation ofosteocytes induces osteoporosis with defective mechanotransduction.
Tatsumi S, Ishii K, Amizuka N, Li M, Kobayashi T, Kohno K, Ito M, Takeshita S, Ikeda K.
Abstract
Bone remodeling is performed by osteoclasts andosteoblasts at the bone surface. Inside of bone is a network of numerousosteocytes, whose specific function has remained an enigma. Here we describe atransgenic mouse model in which inducible and specific ablation of osteocytes isachieved in vivo through targeted expression of diphtheria toxin (DT) receptor.Following a single injection of DT, approximately 70%–80% of the osteocytes,but apparently no osteoblasts, were killed. Osteocyte-ablated mice exhibited fragilebone with intracortical porosity and microfractures, osteoblastic dysfunction, andtrabecular bone loss with microstructural deterioration and adipose tissue proliferationin the marrow space, all of which are hallmarks of the aging skeleton.Strikingly, these 『『osteocyte-less』』 mice were resistant to unloading-inducedbone loss, providing evidence for the role of osteocytes inmechanotransduction. Thus, osteocytes represent an attractive target for thedevelopment of diagnostics and therapeutics for bone diseases, such asosteoporosis.
摘要
眾所周知，骨重構是由成骨細胞和破骨細胞在骨表面進行的，而存在於骨組織內部大量的骨細胞的功能是尚未可知的。在本研究中，通過特異性表達白喉毒素（DT） 受體，我們構建了一種特異性缺失骨細胞的可誘導小鼠模型。單次注射DT，約70%-80%的骨細胞發生消亡，但成骨細胞不受影響。骨細胞消亡的小鼠表現出骨骼老化的特性，即骨脆性增加、骨內部存在孔隙並伴有微小骨折、成骨功能障礙、骨小梁丟失、骨髓間隙超微結構退化和脂肪組織增生。令人驚奇的是，這種「骨細胞減少」的小鼠可以抵抗卸荷導致的股丟失，提示骨細胞在應力傳導過程中扮演重要角色。總之，骨細胞為骨相關疾病如骨質疏鬆症的診斷和治療提供了一個非常有潛力的靶標。
圖一、骨細胞特異性表達DT受體的轉基因小鼠模型的構建
（A）轉基因結構原理圖，（B）RT-PCR檢測靶基因（DT—R）表達情況，可見在骨和牙本質有表達，（C-E）免疫組織化學檢測8周齡轉基因小鼠股骨DT——R表達情況，可見在骨小梁（C）和骨表面（D）的骨細胞中有陽性染色，（E）圖為高倍鏡下的典型陽性細胞。該圖表明轉基因小鼠模型在基因、蛋白層面是構建成功的，且只在骨細胞中特異表達靶基因。
Figure 1.Generation of Transgenic Mice Expressing DT-R Specifically in Osteocytes
(A) Schematicrepresentation of the transgenic construct (pDTR-9.6kb). Diphtheria toxinreceptor (DT-R, dotted box) with poly(A) signals (dark box) was placed underthe control of the 9.6 kb mouse DMP1 promoter (thick line) plus exons 1 and 2(white bars) and intron 1 between them. The arrows indicate the set of primersused for confirming integration of the transgene (one in exon 2 of the DMP1gene, and the other in the DT-R cDNA). (B) RT-PCR analysis of transgene (DT-R)expression in DTR-9.6kb transgenic line #2. RNA for bone and dentin wasprepared from tibiae/femurs and incisors, respectively.(C–E)Immunohistochemical detection of the transgene product, DT-R, by anti-DT-R(hHB-EGF) antibody in osteocytes of trabecular (C) and cortical bone (D) of thefemur of an 8-week-old transgenic mouse. (GP, growth plate; TB, trabecularbone; CB, cortical bone.) Representative osteocytes with prominent staining areindicated by black arrows. A high magnification of a trabecula reveals thatosteoblasts on the surface do not express DT-R (redarrows in [E]).
圖二、轉基因小鼠中骨細胞發生消亡
（A）選取10周齡的雌鼠，WT組注射PBS，Tg組注射50μg/kg劑量的DT，8天後取材，H&E染色股骨皮質，只有注射DT的轉基因小鼠骨皮質有空隙；（B—E）電鏡分析骨細胞形態，來自WT組的正常形態骨細胞（B），來自DT組的異常骨細胞，核凝結（C）、破碎（D）和空泡化（E）；（F）DT注射前/後空泡化（紅色）、凋亡（黃色）和正常（藍色）骨細胞計數分析；（G）DT注射36小時後凋亡染色。
該圖表明注射DT前，轉基因小鼠的骨細胞正常；注射DT後，轉基因小鼠的骨細胞的確發生了消亡。
Figure 2.TargetedAblation ofOsteocytes in Transgenic Mice (A) Ten-week-old malewild-type (WT, upper panels) and transgenic (Tg, lower panels) mice were injectedi.p. with PBS (left panels) or 50 mg/kg body weight DT (right panels), andfemoral cortical bone was stained with hematoxylin and eosin at 8 days post DTtreatment.Note that empty lacunae are observed only in cortical bone oftransgenic mice injected with DT (arrows). Scale bars = 50 mm. (B–E) Osteocytemorphology by electron microscopy showing a normal osteocyte (B), osteocytes withnuclear condensation (C) or fragmentation (D), and an empty lacuna (E) fromwild-type mice (B) or transgenic mice injected with DT (C–E) at 8 days. (F)Numbers of empty lacunae (red), lacunae with apoptotic osteocytes (yellow), andlacunae with normal osteocyte morphology (blue) were counted in femoralcortical bone from WT and Tg miceat 8 days following injection of PBS or DT. Mean values are shown as a percentageof all lacunae (n = 3–6). (G) TUNEL staining (arrows) of femoral sections of WTand Tg mice 36 hr after DT administration.
圖三、骨細胞消亡後產生的短期效應
（A）H&E染色顯示：DT注射8d後，股骨皮質骨出現空隙（箭形指示），且有血管入侵；TRAP（B）、ALP（C）和von Kossa（D）染色，(B)中的箭頭和星號分別表示空腔和內腔。(D)中紅色箭頭和白色箭頭所示的藍色染色分別表示內骨表面和腔內壁上的類骨組織；（E）電子顯微鏡顯示：骨細胞消亡後後出現的微骨折(黃色箭頭)，紅色箭頭表示裂紋中的紅細胞，星號表示空腔；（F）脛骨近端小梁骨處，骨表面的骨樣表面分數(os/bs)分析；（G）骨細胞消融後8天，脛骨近端micro-CT測定骨小梁體積/體積（BV/TV）不變；（H）骨細胞消融後8天，TRAP染色；（I）注射PBS/DT7天後，股骨骨形成的組織形態計量學相關指標—骨表面成骨細胞(ob.s)和骨形成率(Bfr)分析。
Figure 3. Short-Term Effects ofOsteocyte Ablation (A) Histological analysis of hematoxylin-andeosin- stainedsections of femurs from 10- week-old WT (left) and Tg (right) mice at 8 daysafter a single injection of DT. Note the empty lacunae (arrows) andintracortical cavities with vascular invasion. (BV in inset, blood vessel.)(B–D) TRAP (B), ALP (C), and von Kossa (D) staining of femurs of WT (left) andTg (right) mice injected with DT. Arrows and the asterisk in (B) indicate emptylacunae and intracortical cavities, respectively. The blue staining shown bythe red arrows and white arrows in (D) indicates osteoid on the endostealsurface and on the wall of intracorticalcavities, respectively. (E) The presence of microfractures (yellow arrows)following osteocyte ablation as revealed by electron microscopy. Red arrowsindicate red blood cells in the cracks. The asterisk indicates an empty lacuna.Magnification 1000x. (F) Osteoid surface fraction per bone surface (OS/BS)measured at the trabecular bone of the proximal tibia. In this and all otherfigures, error bars indicate ±SEM. *p < 0.05 (n = 5) (G) Trabecular bonevolume/tissue volume (BV/TV) in the proximal tibia as determined by micro-CTwas unchanged at 8 days following osteocyte ablation. (H) TRAP staining of thetrabecular bone of WT (left) and Tg (right) mice at 8 days after DTadministration. (I) Histomorphometric indices of bone formation in the femur at7 days after injection of DT or PBS in 10-week-old WT or Tg mice. Osteoblastsurface (Ob.S) and bone formation rate (BFR) were corrected for bone surface(BS). MAR, mineral apposition rate; Mlt, mineralization lag time. **p < 0.01(n = 3 per group).
圖四、定量RT—PCR分析骨細胞相關的分子標記物表達情況。
結果顯示：DT-R、DMP1、FGF-23、SOST、MEPE、Phex、E11/gp38顯著降低，RunX2無明顯差異。
Figure 4. Quantitative RT-PCRAnalysis of Molecular Markers of Bone Cells After the bone marrow had beenflushed out, RNA was extracted from the femurs and tibiae of 10-week-oldDTR-9.6kb transgenic mice injected with PBS (white bars) or DT (black bars) at2 days. Gene expression was assessed by real-time PCR using a LightCyclersystem and normalized to EF-1a mRNA. MEPE, matrix extracellularphosphoglycoprotein; Phex, phosphate-regulating gene with homologies to endopeptidaseson the X chromosome; RANKL, receptor activator of NF-kB ligand; OPG,osteoprotegerin; ALP, alkaline phosphatase; OC, osteocalcin. **p < 0.01; *p< 0.05 (n = 3 mice per group).
圖五、骨細胞消融後產生的長期效應
（A和B）18周齡的小鼠，單次注射PBS/DT 40天後，H&E染色顯示：骨細胞消融後皮質骨的變薄和脂肪組織在骨髓間隙的積聚；（B）圖為(A)圖中的部分皮質骨，箭頭指示內部孔隙；(C和D)注射PBS/DT 40d後，股骨皮質內孔隙面積(po.ar/ct.ar)(C)和骨髓脂肪體積(%脂肪體積/骨髓體積)(D)的分析；(E、F和G)micro-CT掃描結果分析及三維重構；（H）注射40d和90d後骨強度分析。
Figure 5. Osteoporotic Changes asLong-Term Consequences of Osteocyte Ablation (A and B) Representative tibial sectionsof 18-week-old WT and Tg mice at 40 days following a single DT administration.Note the thinning of the cortical bone and accumulation of adipose tissue inthe marrow space following osteocyte ablation. Scale bars = 200 mm. Part of thecortical bone in (A) is highlighted in (B) to illustrate intracortical porosity(arrows). (C and D) Intracortical porosity area per cortical area (Po.Ar/Ct.Ar)(C) and marrow adiposity (% fat volume/marrow volume) (D) of femurs at 40 daysafter DT administration. **p < 0.01 (n = 5–6 per group). (E and F) Representativemicro-CT images of lumbar vertebrae of WT and Tg mice at 40 days following DTadministration (E) with microstructural parameters (BV/TV, 3D bone volumefraction per tissue volume; Conn-Dens., connectivity density; SMI, structuremodel index; Tb.Th, trabecular thickness) derived from micro-CT analysis (F).**p < 0.01 (n = 5 per group). (G and H) Recovery of bone volume (G) and bonestrength (H) along with osteocyte regeneration. Trabecular bone volume and bonestrength were determined by micro-CT and the four-point bending test,respectively, on the femurs of transgenic mice injected with PBS (white bars)or DT (black bars) at 40 and 90 days. *p < 0.05; **p < 0.01 (n = 5 per group).
圖六、骨細胞缺失可以抵抗卸荷引起的骨量丟失
（A）micro-CT掃描時間點示意圖，尾吊模型模擬卸荷作用，正常生活的即為對照，在尾吊前一天注射DT；（B）尾吊7天後脛骨幹骺端三維骨體積的變化情況；（C）破骨細胞數(n.oc/bs)和骨形成率(BfR/bs)在尾吊和不尾吊小鼠中的情況分析；（D）定量RT-PCR分析RANKL、OPG和SOST表達情況。
Figure 6. Osteocyte-Ablated MiceAre Resistant to Unloading-Induced Bone Loss (A) Experimental schedule withrepresentative micro-CT images. Eighteen-week-old WT and Tg mice were subjectedto skeletal unloading by tail suspension (TS) for 7 days. Mice on the ground(Gr) served as controls. DT was administered 1 day prior to the initiation ofTS (_1). (B) Changes in 3D bone volume at the tibialmetaphysis following 7 day unloading by tail suspension (black bars). Mice onthe ground (white bars) served as controls. **p < 0.01 (n = 5 per group). (C)Number of osteoclasts (N.Oc/BS) and bone formation rate (BFR/BS) in TS (blackbars) and ground control (white bars) mice. *p < 0.05 (n = 5 per group). (D)Quantitative RT-PCR analysis. RNA was extracted from tibiae and femurs of WTand DTR-9.6kb Tg mice injected with DT (red bars) or PBS (pale green bars) at 3days following the initiation of TS, after bone marrow had been flushed out.Transgenic mice on the ground (Gr) served as controls (white and black bars).*p < 0.05; **p < 0.01 (n = 3 per group).
圖七、恢復負荷後，骨細胞缺失不能引起骨量增加。
小鼠卸荷—尾吊處理7天後再去除尾吊，14天以後觀察，（A）圖示意4個分組間的不同處理及三維重構情況；（B和C）不同分組間組織形態計量學指標(Bs)分析，小鼠脛骨幹骺端三維骨體積的變化情況(B)、破骨細胞數目(n.oc)和骨形成率(BfR)；（D）根據本研究的結果，在不同應力條件下骨細胞的作用不同。在正常機械負荷條件下，骨細胞具有維持破骨細胞(Oc)的骨吸收，成骨細胞(Ob)礦化的功能。當骨細胞發生消亡時，骨吸收異常升高與礦化受損。當應力缺失時，骨細胞刺激骨吸收、抑制骨形成，短期內即可引起明顯的骨丟失和微結構惡化；而若應力缺失且骨細胞消融時，這些變化就不會發生，骨細胞可以抵抗應力缺失引起的骨質疏鬆。不過，而重新加載應力後，據本研究的結果提示，破骨細胞(Occ)和成骨細胞(Ob)對再負荷刺激做出的反應是可以繞過骨細胞，此時骨細胞是非必須的，恢復應力刺激後可以逆轉骨吸收的增加和骨形成的抑制。
Figure 7. Absence of OsteocytesDoes Not Affect Bone Gain during Reloading (A) Experimental schedule forreloading after tail suspension (TS/Gr) and representative micro-CT images. Eighteen-week-oldWT and DTR-9.6kb Tg mice were subjected to TS for 7 days and were then leftambulatory for the following 14 days. DT (4) or PBS as vehicle (3) was injectedbefore initiation of reloading so that osteocytes were ablated specificallyduring the reloading period (4). Mice on the ground throughout the experimentalperiod (1) and mice subjected to TS only (2) served as controls. (B and C)Changes in 3D bone volume at the tibial metaphysis of WT and Tg mice with orwithout DT administration (B) and histomorphometric indices for osteoclastnumber (N.Oc) and bone formation rate (BFR) corrected for bone surface (BS) (C)after reloading (TS/Gr). **p < 0.01; *p < 0.05 (n = 5 per group). (1)–(4)correspond to the four experimental groups shown in (A). (D) Proposed roles ofosteocytes under different mechanical conditions. Based on the current results,under normal loading conditions (Mechanical input +), osteocytes function tokeep osteoclastic (Oc) bone resorption in check and to maintain mineralizationby osteoblasts (Ob). Thus, when osteocytes are ablated, aberrantly elevatedbone resorption with impaired mineralization takes place. In response tounloading (Mechanical input _), osteocytes executethe stimulation of bone resorption and suppression of bone formation, resultingin marked bone loss and microstructural deterioration in a short period. Thus,when osteocytes are ablated specifically during tail suspension, those changesdo not take place, and bone is resistant to disuse-induced atrophy. However,based on the results of the reloading experiments following unloading(Mechanical input _/+), it is suggested that osteocytes are dispensable forthis recovery phase and that osteoclasts (Oc) and osteoblasts (Ob) respond tothe reloading stimulus, bypassing osteocytes, with reversal of elevated boneresorption and release from suppressed bone formation, respectively.
文末驚喜
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