【作者】 杨阳；

【导师】 赵新明；

【作者基本信息】 河北医科大学 ， 影像医学与核医学（专业学位）， 2019， 博士

【摘要】 第一部分^99mTc标记HER2亲合体Z_HER2:V2制备及体外结合特性的研究目的本研究将HER2亲合体Z_HER2:342的N端用-AEN取代-VEN,并对C端用四个氨基酸-G（Gly）GGC（Cys）作为螯合剂修饰,形成新的HER2亲合体Z_HER2:V2。应用配体交换法将^99mTc标记该亲合体,制备亲合体分子影像探针^99mTc-Z_HER2:V2,分析其在体外与HER2受体的结合特性。方法应用Fmoc/tBu多肽固相合成法合成Z_HER2:V2（序列为:AENKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAE AKKLNDAQ）,并在其C末端连接4个氨基酸（-GGGC）,形成一个类似N₃S结构的,能与^99mTc进行强力螯合的短肽,再利用配体交换法标记^99mTc。利用反相高效液相色谱法（reverse-phase high performance liquid chromatography,RP-HPLC）测定分子探针^99mTc-Z_HER2:V2的标记率与放射化学纯度。分析分子探针^99mTc-Z_HER2:V2的体外稳定性,以及在体外与HER2高表达人卵巢癌SKOV3细胞的结合力及滞留率。同时,在体外应用过量未标记^99mTc的Z_HER2:V2对HER2高表达人卵巢癌SKOV3细胞HER2受体进行预阻断,与未阻断（未饱和）组进行对照,探究分子探针^99mTc-Z_HER2:V2与HER2受体结合的靶向特异性。结果分子探针^99mTc-Z_HER2:V2的放射峰是单峰,放射峰保留时间约为12min,标记20min后标记率为98.99%±0.99%（n=6）。^99mTc-Z_HER2:V2与生理盐水及人新鲜血清混合后在8h内的放化纯均在96%以上,且放射峰位置均较稳定。^99mTc-Z_HER2:V2在体外与HER2高表达的人卵巢癌SKOV3细胞具有较高的结合率,24h最高,结合率为6.15%±0.18%。^99mTc-Z_HER2:V2在细胞内滞留率相对较高,最高可达75.26%±3.25%,经过24h后,仍有35.16%±11.23%滞留在细胞中,说明该分子探针可被HER2高表达的人卵巢癌SKOV3细胞高摄取,并能长时间滞留在细胞中。随着时间延长细胞膜结合率整体呈曲线下降趋势,但经过24小时后,仍有近70%的^99mTc-Z_HER2:V2结合在细胞膜上。这表明^99mTc-Z_HER2:V2可与细胞膜上的HER2受体结合并转入细胞内,但这是一个很缓慢的过程。此外,分别加入500倍过量和1000倍过量未标记的Z_HER2:V2对人卵巢癌SKOV3肿瘤细胞HER2受体进行阻断,分子探针与人卵巢癌SKOV3细胞的结合率随阻断倍数的增加而减小,分别为1.33%±0.21%和0.94%±0.13%,表明分子探针^99mTc-Z_HER2:V2与HER2受体的结合具有特异性。第二部分^99mTc标记HER2亲合体Z_HER2:V2体内分布及显像研究目的本研究将对分子探针^99mTc-Z_HER2:V2在HER2高表达人卵巢癌SKOV3荷瘤裸鼠进行体内分布的研究,同时对该分子探针在HER2高表达人卵巢癌SKOV3荷瘤裸鼠的显像,并与HER2低表达人乳腺癌MCF-7荷瘤裸鼠的显像进行对比研究。方法应用贴壁法培养HER2高表达的人卵巢癌SKOV3细胞以及HER2低表达的人乳腺癌MCF-7细胞。裸鼠右前肢外侧皮下接种5×10⁶个人卵巢癌SKOV3细胞或人乳腺癌MCF-7细胞,制备HER2高表达和低表达的移植瘤动物模型。3⁴周后待移植瘤直径长至1.5².0cm,选取瘤体饱满、无坏死、大小无显著性差异的荷瘤裸鼠模型纳入实验。再选取16只移植瘤大小无明显差异的人卵巢癌SKOV3细胞荷瘤裸鼠,随机分成4组,每只荷瘤裸鼠均经由尾静脉注射分子探针^99mTc-Z_HER2:V2。分别于注药后的第1h、2h、4h和6 h随机取1组裸鼠,麻醉后经眼静脉取血,并颈椎脱臼处死。解剖出心脏、肝脏、脾脏、肺、肾脏、胃、小肠、脑、肌肉、骨骼及肿瘤组织,分别称重,并且测定每个组织的放射性计数。计算每克组织的百分注射率即%ID/g,及肿瘤与对侧肌肉组织的%ID/g比值（tumor to muscle,T/M）。分析随时间变化分子探针^99mTc-Z_HER2:V2在人卵巢癌SKOV3细胞荷瘤裸鼠体内的代谢情况,以及荷瘤裸鼠体内不同器官（或组织）中的放射性分布情况。随机抽取移植瘤大小无明显差异的5只HER2高表达的人卵巢癌SKOV3细胞荷瘤裸鼠及5只HER2低表达的人乳腺癌MCF-7细胞荷瘤裸鼠,将分子探针^99mTc-Z_HER2:V2经由尾静脉注射至荷瘤裸鼠体内。分别于注药后1h、2h、4h、6h和8 h进行显像,观察分子探针^99mTc-Z_HER2:V2在移植瘤内的浓聚情况,分别计算荷瘤裸鼠在不同时间点移植瘤与同等大小的对侧区域的放射性计数比值,即T/NT比值。对^99mTc-Z_HER2:V2在HER2高表达的人卵巢癌SKOV3细胞荷瘤裸鼠及HER2低表达的人乳腺癌MCF-7细胞荷瘤裸鼠的体内显像差异进行对比分析。另取5只人乳腺癌SKOV3细胞荷瘤裸鼠模型,每只荷瘤裸鼠经由尾静脉注射过量未标记^99mTc的HER2亲合体Z_HER2:V2使移植瘤组织HER2的受体封闭,5min后再注射分子探针^99mTc-Z_HER2:V2,按上述显像方法于注药后4h进行显像,对阻断组与非阻断组荷瘤裸鼠移植瘤部位放射性浓聚情况进行对比观察,并分析分子探针^99mTc-Z_HER2:V2与人卵巢癌SKOV3细胞移植瘤的HER2受体的体内特异性靶向结合特性。探讨分子探针^99mTc-Z_HER2:V2在活体内探测病灶HER2表达的应用价值。结果1.HER2高表达的人卵巢癌SKOV3细胞移植瘤对^99mTc-Z_HER2:V2的体内摄取随注射时间延长而升高,且在6h达到高峰,为9.38±1.22%ID/g,表明^99mTc-Z_HER2:V2在人卵巢癌SKOV3细胞荷瘤裸鼠的移植瘤组织中的分布随时间增长而逐渐增加。以6h为例,移植瘤部位与对侧肌肉部位T/M值约为14.31±3.16,表明移植瘤对分子探针^99mTc-Z_HER2:V2摄取较高。除移植瘤以外,分子探针^99mTc-Z_HER2:V2在肾脏中的放射性分布最高,而肝脏中的放射性分布明显比肾脏低,在心脏、骨骼、肺脏和肌肉组织内放射性分布非常低,血液内的放射性亦较低,表明分子探针^99mTc-Z_HER2:V2大部分经肾脏代谢,而肝脏摄取率低,且探针血液清除快。2.HER2高表达人卵巢癌SKOV3细胞荷瘤裸鼠经由尾静脉注射分子探针^99mTc-Z_HER2:V2后1h显像时即可看到移植瘤部位显影,并且随着显像时间延长,放射性浓聚程度逐渐增加。除肿瘤以外,双侧肾脏和膀胱有明显的放射性浓聚,然而在甲状腺、肝脏等部位却始终未见明显的放射性浓聚,利用ROI技术测得的T/NT值以8h最高为14.13±1.22,这样的结果能够证明分子探针^99mTc-Z_HER2:V2体内稳定性较好,而且主要经肾脏排泄。3.对HER2高表达的人卵巢癌SKOV3细胞荷瘤裸鼠提前注射过量未标记^99mTc的Z_HER2:V2,使HER2受体封闭后再行SPECT显像,未看到移植瘤组织明显摄取分子探针^99mTc-Z_HER2:V2,这表明未标记的Z_HER2:V2可将人卵巢癌SKOV3细胞移植瘤组织的HER2受体封闭,分子探针^99mTc-Z_HER2:V2与人卵巢癌SKOV3细胞荷瘤裸鼠的移植瘤在体内具有HER2靶向结合特性。4.对HER2低表达的人乳腺癌MCF-7细胞荷瘤裸鼠注射^99mTc-Z_HER2:V2后,分别于不同时间点显像,可见只有双侧肾脏及膀胱可见明显的放射性浓聚,各时间点移植瘤所在部位均未见明显放射性浓聚,T/NT比值均明显低于HER2高表达的人卵巢癌SKOV3细胞荷瘤裸鼠,各时间点均有显著性差异（t值分别为23.595,38.678,14.44,12.00,12.80,P值分别为0.000,0.000,0.000,0.000,0.000）。再次证明分子探针^99mTc-Z_HER2:V2在活体内可被HER2高表达的人卵巢癌SKOV3细胞移植瘤摄取。第三部分^99mTc标记HER2亲合体Z_HER2:V2显像监测HER2阳性肿瘤早期疗效的研究目的本研究将探讨分子探针^99mTc-Z_HER2:V2 SPECT显像对化疗联合赫赛汀靶向治疗HER2高表达肿瘤疗效评价的价值。方法实验随机共分两组,每个治疗组荷瘤裸鼠3只。治疗组1（化疗组）:分别于治疗的第1d、4d、8d、11d,腹腔注射顺铂3mg/kg,紫杉醇20mg/kg;治疗组2（化疗联合靶向治疗组）:于治疗的第1d、4d腹腔注射顺铂3mg/kg,紫杉醇20mg/kg,并于第8d,腹腔注射赫赛汀40mg/kg。治疗期间,每3d测定荷瘤裸鼠的体重、移植瘤体积。利用游标卡尺测量移植瘤的最长径（L）及最短径（D）,移植瘤体积按如下公式计算:肿瘤体积=L×D²×1/2。分别于治疗前、治疗第8d、15d对各组荷瘤裸鼠进行^99mTc-Z_HER2:V2SPECT显像。经尾静脉注射分子探针^99mTc-Z_HER2:V24h后上机检查,取仰卧位扫描。观察各组人卵巢癌SKOV3细胞荷瘤裸鼠移植瘤的放射性浓聚情况。同时利用感兴趣区技术（Region of Interest,ROI）,分别对两个治疗组荷瘤裸鼠移植瘤与同等大小对侧区域的放射性计数的比值进行计算,即T/NT（target to nontarget）比值,并对两个治疗组进行对比分析。于治疗的第15d SPECT显像结束后,处死各组荷瘤裸鼠。取出移植瘤组织,观察移植瘤的大体形态,并进行免疫组化分析,观察移植瘤组织经治疗后对HER2的表达情况。最后分析分子探针^99mTc-Z_HER2:V2SPECT显像评价化疗联合赫赛汀治疗HER2高表达肿瘤疗效的临床价值。结果治疗结束后,两个治疗组荷瘤裸鼠体重经统计学分析,无显著性差异（F=0.579,P=0.689;F=0.462,P=0.762）,表明化疗或者化疗联合靶向治疗对荷瘤裸鼠的体重增长与否无明显影响。在前7d的治疗中,两个治疗组荷瘤裸鼠的移植瘤体积逐渐增大,治疗7d结束后,两治疗组荷瘤裸鼠移植瘤体积无显著性差异（t=0.165,P=0.877>0.05）;在后8d的治疗中,治疗组2中的荷瘤裸鼠的移植瘤体积逐渐缩小,而治疗组1的移植瘤体积仍然继续增大。治疗结束后,治疗组2中的荷瘤裸鼠的移植瘤体积明显小于其治疗组1（t=18.318,P=0.000<0.05）。这样的结果可以说明:化疗在联合应用HER2靶向药物后,可缩小移植瘤的体积,以达到治疗最佳的目的。治疗组1及治疗组2的HER2高表达人卵巢癌SKOV3细胞荷瘤裸鼠分别于治疗前、治疗后第8d、15d进行^99mTc-Z_HER2:V2SPECT显像。治疗前,各组HER2高表达人卵巢癌SKOV3细胞裸鼠移植瘤部位均可见放射性浓聚。经治疗后,治疗组1移植瘤部位放射性浓聚先减轻（治疗前7d）后增强（治疗后8d）;治疗组2移植瘤部位放射性浓聚逐渐减轻。治疗前,两个治疗组荷瘤裸鼠移植瘤部位T/NT比值无明显统计学差异（t=1.518,P=0.204>0.05）;经前7d治疗后,治疗组1和治疗组2荷瘤裸鼠肿瘤部位T/NT比值明显减小。在后8d治疗中,治疗组2荷瘤裸鼠移植瘤部位T/NT比值继续减小,治疗组1荷瘤裸鼠移植瘤部位T/NT比值较治疗前7d有所增加。当治疗结束后,两个治疗组荷瘤裸鼠移植瘤部位T/NT比值相比,有统计学差异（t=7.962,P=0.001<0.05）。且治疗组2荷瘤裸鼠移植瘤部位T/NT比值低于治疗组1。治疗组1及治疗组2的HER2高表达人卵巢癌SKOV3细胞荷瘤裸鼠经治疗后移植瘤组织的免疫组织化学分析:治疗组1可见中等程度的棕黄色染色;治疗组2可见仅有极少程度的棕黄色染色。结果表明,化疗联合HER2靶向治疗可以有效的降低HER2的表达水平。同时也证明了分子探针^99mTc-Z_HER2:V2可以作为监测肿瘤治疗早期疗效的显像剂。结论1.本研究中成功制备了核素^99mTc标记的HER2亲合体Z_HER2:V2。分子探针^99mTc-Z_HER2:V2的标记率高,标记方法简单易行,无需纯化;其体外稳定性好,可在体外与HER2高表达的人卵巢癌SKOV3细胞的HER2受体特异性结合,并且长时间滞留在细胞中,⁹9m9m Tc-Z_HER2:V2是一个很有潜力的分子影像探针。2.分子探针^99mTc-Z_HER2:V2的体内分布特性良好,HER2高表达的人卵巢癌SKOV3细胞荷瘤裸鼠的移植瘤部位摄取明显,大部分经肾脏排泄,肝脏摄取少,血液清除快,图像质量较高。分子探针^99mTc-Z_HER2:V2在体内与人卵巢癌SKOV3细胞荷瘤裸鼠移植瘤的HER2受体具有良好的靶向结合特性。分子探针^99mTc-Z_HER2:V2在活体内可探测病灶的HER2表达。3.赫赛汀联合化疗比单一的化疗对HER2高表达的人卵巢癌SKOV3移植瘤更具有明显治疗疗效,并且能显著缩小肿瘤体积,明显下调肿瘤组织HER2的表达水平。分子探针^99mTc-Z_HER2:V2可监测其治疗效果。更多还原

【Abstract】 Part one:Preparation and in vitro study of ^99mTc labeled Z_HER2:V2Objective The research aimed to replace-VEN with-AEN at the N-terminal for affibody Z_HER2:342ER2:342 of HER2 and to use four amino acids-G（Gly）GGC（Cys）as chelating agent for decoration at the C-terminal.Direct labeling method was used to label radionuclide ^99mTc,to prepare affibody molecule imaging probe ^99mTc-Z_HER2:V2,and to analyze its HER2 binding characteristics in vitro.Method Fmoc/tBu solid-phase synthesis is used to synthesize Z_HER2:V2（sequence:AENKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAE AKKLNDAQ）and four amino acids（-GGGC）are connected at C-terminal so as to form an oligopeptide which can be strongly chelate with the structure being similar to N₃S.In addition,ligand-exchange chromatography is used to label ^99mTc.Reverse-phase high performance liquid chromatography（RP-HPLC）is used to determine labeling rate and radiochemical purity of the probe.Stability in vitro and its binding capacity and retention rate in vitro with HER2 over-expression ovarian cancer SKOV3 cells are analyzed.Meanwhile,comparison with non-interruption（unsaturation）group which wasused excessively unlabelled Z_HER2:V2ER2:V2 to pre-interrupt HER2 receptors of SKOV3cells in vitro,so as to research binding characteristics of the molecular probe.Result Radioactive peak of molecular probe ^99mTc-Z_HER2:V2 is a single peak,retention time of radioactive peak is 12min,andlabeling rate is98.99%±0.99%（n=6）.Radiochemical rate is more than 96%,not only^99mTc-Z_HER2:V2 mixed with normal saline but also mixed with fresh serum within 8h;moreover,position of radioactive peak is stable.^99mTc-Z_HER2:V2inHER2 over-expression ovarian cancer SKOV3 cells has higher binding rate which is the highest at the 24h with binding rate of 6.15%±0.18%in vitro.^99mTc-Z_HER2:V2 has relatively higher retention rate in the cells,which can be75.26%±3.25%at the highest.In case there is about 35.16%±11.23%of^99mTc-Z_HER2:V2left in the cells after 24h,it indicates that the molecular probe can be highly taken in by HER2 over-expression SKOV3 cells and can be left in the cells for a long time.Binding rate of cells membrane is in curve decline trend with time extension.However,there is about 70%of ^99mTc-Z_HER2:V2 are bound on cells membrane after 24h,which indicates that ^99mTc-Z_HER2:V2 can be bound with HER2 receptors on the cells membrane and can be transferred into the cells,but it is a slow process.In addition,after 500 times and 1000 times of excessive unlabelled Z_HER2:V2 are respectively added to SKOV3 cells for interruption,binding rate between molecular probe and SKOV3 cells will be decreased with increase of interruption times,which is respectively1.33%±0.21%and 0.94%±0.13%,and indicates that specificity binding characterize of molecular probe ^99mTc-Z_HER2:V2.Part two:The distribution and imaging studies of ^99mTc-Z_HER2:V2 in vivoObjective Body distribution of ^99mTc-Z_HER2:V2 in nude mice bearing the HER2 over-expression SKOV3 xenografts was researched;meanwhile,comparative research was made for the molecular probe in SKOV3 nude mice（HER2 over-expression）and that in MCF-7 nude mice of（HER2 low expression）.Method Adherence method is used to culture HER2 over-expression ovarian cancer SKOV3 cells and HER2 low expression breast cancer MCF-7cells.5×10⁶ ovarian cancer SKOV3 cells or MCF-7 cells are subject to external subcutaneous inoculation of right fore limb for nude mice so as to prepare HER2 over-expression and low expression xenografts animal model.The nude mice with full,no necrosis,and no significant differences in size should be selected to be incorporated in the experiment when tumor diameter is grown to 1.5².0cm 3 or 4 weeks later.Sixteen SKOV3 nude mice with the no obvious differences tumor size should be selected,which should be randomly divided into 4 groups.^99mTc-Z_HER2:V2ER2:V2 should be injected into each nude mice through caudal vein.A group of nude mice should be randomly taken at the 1^st hour,the 2^nd hour,the 4^th hour,and the 6^th hour after injection.Then,they should be subject to vena ophthalmica so as to collect blood and should be killed with the way of cervical dislocation.Heart,liver,spleen,lung,kidney,stomach,small intestine,cerebrum,muscle,skeleton,and tumor tissue should be taken so as to respective weigh quality.Moreover,its radioactivity counts should be determined;percentage injection rate which is%ID/g of tissue per gram and ratio between tumor and muscular tissue（tumor to muscle,T/M）should be calculated.Moreover,metabolism of^99mTc-Z_HER2:V2 in nude mice with ovarian cancer SKOV3 cells and radioactive distribution in different organs or tissues should be analyzed.Five nude mice with HER2over-expression of SKOV3 ovarian cancer cells and five nude mice with HER2 low expression of MCF-7 breast cancer should be randomly selected;^99mTc-Z_HER2:V2 should be injected into the nude mice through caudal vein.SPECT imaging should be taken 1^st hour,the 2^nd hour,the 4^th hour,the 6^th hour and the 8^th hour after injection.Later after injection so as to observe concentration of the probe in the tumors and to respectively calculate theT/NT ratio between tumor and contralateral area with the same sizeat different time point.Comparative analysis of SPECT imaging for ^99mTc-Z_HER2:V2 in nude mice with HER2 over-expression of SKOV3 ovarian cancer cells and nude mice with HER2 low expression of MCF-7 breast cancer cells.In addition,5models ofnude mice with SKOV3 ovarian cancer are taken;each nude mice should be subject to injection of excessively unlabelled HER2 affibody Z_HER2:V2ER2:V2 so as to close the HER2 receptors of tumor tissue through caudal vein.^99mTc-Z_HER2:V2 should be injected 5min later.SPECT imaging should be conducted 1^st hour,the 2^nd hour,the 4^th hour,the 6^th hour and the 8^th hour later after injection according to above-mentioned SPECT imaging method so as to make comparative observation of radioactivity concentration for tumor site of nude mice in the interruption group and the non-interruption group,and analyzed the binding characteristics of ^99mTc-Z_HER2:V2in vivo.The value of HER2 expression detection in vivo for using molecular probe ^99mTc-Z_HER2:V2should be discussed.Result1. In vivo,the uptake of HER2 over-expression SKOV3 tumor to^99mTc-Z_HER2:V2ER2:V2 will be risen with injection time extension and will reach the peak which is 9.38±1.22%ID/g at the time of 6h.It indicates that^99mTc-Z_HER2:V2ER2:V2 distributed in tumor of SKOV3 nude mice will be gradually increased with time extension.Such as 6h,T/Mratio between tumor and contralateral muscle is 14.31±3.16,which indicates that tumor has high uptakeof molecular probe.In addition,^99mTc-Z_HER2:V2 has the highest radioactive distribution in the kidneys.Meanwhile,radioactive distribution in the liver is obviously lower than kidney,which indicates that most of the molecular probe is subject to metabolism through kidney;liver has a low uptake rate;radioactive distribution in heart,skeleton,lung,and muscular tissue is low.Radioactivity in the blood is low,which indicates that the molecular probe can be quickly eliminate from blood.2.Tumor can be seen in SPECT imaging of 1h later after SKOV3nude mice are subject to intravenous injection of probe^99mTc-Z_HER2:V2.Moreover,radioactivity concentration will be gradually increased with imaging time extension.In addition,there is obvious radionuclide concentration in both kidneys and bladder,while there is no obvious radionuclide concentration in liver,thyroid,and other positions.The highest T/NT ratio,which is 14.13±1.22 at 8h measured by ROI technology.2.SKOV3 nude mice should be injected with excessively unlabelled Z_HER2:V2 in advance so as to conduct SPECT imaging after HER2 receptorsare closed.Obvious uptake of tumor tissue is not seen,which indicates that unlabelled Z_HER2:V2 can close HER2 receptors of SKOV3 tumor;^99mTc-Z_HER2:V2 have specificity binding characteristics in vivo.3.SPECT imaging for MCF-7nude mice at different time points after injection of ^99mTc-Z_HER2:V2 should be conducted.Radionuclide concentration can be obviously seen in kidney and bladder;there is no obvious imaging of tumor at different time points.T/NT ratio is obviously lower than SKOV3nude mice,and there is significant difference at different time points（t value is respectively 23.595,38.678,14.44,12.00,and 12.80,while P value is respectively 0.000,0.000,0.000,0.000,and 0.000）.This result can be included ^99mTc-Z_HER2:V2could be uptook by HER2 over-expression’s tumors in vivo.Part three:The study of ^99mTc-Z_HER2:V2 SPECT imaging to monitoring the early efficacy in HER2 positive tumors’treatmentObjective This study will investigate the value of molecular probe^99mTc-Z_HER2:V2 SPECT imaging in the evaluation of the efficacy of chemotherapy combined with herceptin in the treatment of HER2over-expression tumors.Method The nude miceare divided into two groups.Each treatment group has 3 nude mice.Treatment group 1（chemotherapy group）:they should be respectively subject to intraperitoneal injection of 3mg/kg cis-platinum and20mg/kg paclitaxel at the 1d,the 4d,the 8d,the 11d of treatment;Treatment group 2（chemotherapy allies anti-HER2 group）:they should be respectively subject to intraperitoneal injection of 3mg/kg cis-platinum and 20mg/kg paclitaxel at the 1d and the 4d of treatment and should be subject to intraperitoneal injection of 40mg/kg trastuzumab at the 8d of treatment.Weight and tumor volume of nude mice should be determined per 3d during the treatment period.Vernier caliper should be used to measure the longest diameter（L）and the shortest diameter（D）of tumor.Tumor volume should be calculated according to the following formula:tumor volume=L×D²×1/2.Each group of nude mice should be respectively subject to ^99mTc-Z_HER2:V2SPECT imaging before treatment and the 8d and the 15d later after treatment.The supine position imaging should be taken after molecular probe^99mTc-Z_HER2:V2was subject to tail intravenous injection 4h later.Radioactivity concentration of tumor for each group of SKOV3 nude mice should be observed.Meanwhile,region of interest（ROI）should be used to respectively calculate T/NT（target to nontarget）ratio and to make comparative analysis of the treatment group.All groups of nude mice should be killed after termination of SPECT imaging at the 15d of treatment;tumor tissue should be taken out;general form of tumor tissue should be observed so as to make immunohistochemical analysis.HER2 expression after treatment of tumor tissue should be observed.Finally,value of ^99mTc-Z_HER2:V2SPECT imaging in therapeutic evaluation of HER2 over-expression tumor for chemotherapy combined with herceptin treatment should be observed.Result Weight of nude mice in two treatment groups will be subject to statistical analysis after termination of treatment with no significant differences（F=0.579,P=0.689;F=0.462,P=0.762）,which indicates that the weight of nude mice was not influence by chemotherapy or chemotherapy allies anti-HER2.Tumor volume of nude mice in the two treatment groups is gradually increased in the first 7d of treatment,and has not significant differences（t=0.165,P=0.877>0.05）after termination of 7d treatment.Tumor volume of nude mice in the treatment group 2 is gradually shrunk in the last8d of treatment,while in the treatment group 1 is continued to be increased.Tumor volume of nude mice in the treatment group 2 is obviously smaller than treatment group 1（t=18.318,P=0.000<0.05）.The result indicates that chemotherapy can be used to shrink volume of tumor after being combined with the antiHER2 drug so as to reach the purpose of treatment.Treatment group 1 and treatment group 2 can be subject to ^99mTc-Z_HER2:V2ER2:V2 SPECT imaging before treatment and on the 8d,15d after treatment.Radionuclide concentration can be seen in the tumor of SKOV3nude mice in different groups before treatment.Radionuclide concentration of tumor position in treatment group 1 can be firstly reduced（the first 7d of treatment）and can be enhanced（the last 8d of treatment）after treatment.Radionuclide concentration of tumor in treatment group 2 can be gradually reduced.T/NT ratio of tumor for nude mice in the two groups of treatment has no obvious statistical differences（t=1.518,P=0.204>0.05）.T/NT ratio of tumor for nude mice in the treatment group 1 and the treatment group 2 is obviously decreased.T/NT ratio of tumor for nude mice in the treatment group 2 is continued to be decreased in the last 8d treatment;T/NT ratio of tumor for nude mice in the treatment group 1 is increased than that in the first 7d of treatment.T/NT ratio of tumor for nude mice in the two treatment groups are compared after termination of treatment,which has statistic differences（t=7.962,P=0.001<0.05）.In addition,T/NT ratio of tumor position for nude mice in the treatment group 2 is lower than that in the treatment group1.Iimmunohistochemical analysis of tumor tissue after treatment of SKOV3nude mice with HER2 over-expression in treatment group 1,and treatment group 2:brown dye to a middle extent can be seen in the treatment group 1;brown dye to the rare extent can be seen in the treatment group 2.The result indicates that combined anti HER2 treatment with chemotherapy can be used to effectively reduce HER2 expression’s level.Meanwhile,molecular probe ^99mTc-Z_HER2:V2 is proved to be as an excellentmolecular probe to monitor the early efficacy of tumor treatment.Conclusion:1.The research successfully to prepare HER2 small molecular targeted binding protein Z_HER2:V2labelled by Radionuclide^99mTc.The labeling method is simple and feasible with a high labeling rate and a good stability in vitro,which is not required to be purified.It can be specificity bound with HER2over-expression ovarian cancer SKOV3cellsin vitro,and can be retention in the cells for a long time,thus it is a potential molecular imaging probe.2.The molecular probe ^99mTc-Z_HER2:V2 has some advance in vivo,such as,good bio-distribution;tumor uptake of SKOV3nude mice is obvious,most of which are excreted through kidneysand with less uptake in the liver,quick blood elimination,and good image quality.^99mTc-Z_HER2:V2 has good specificity binding characteristics ofHER2receptors in vivo.Detection HER2 expression of molecular probe ^99mTc-Z_HER2:V2 in vivo nidus is feasible.3.Treatment of chemotherapy combined with trastuzumab has more obviousefficacytoHER2over-expressionovariancancer SKOV3xenograftstumor than that of single chemotherapy.In addition,it can significantly shrink tumor volume and can obviously reduce HER2 expression level of tumor tissue.So that thetherapeutic efficacy could be monitored by molecular probe ^99mTc-Z_HER2:V2.更多还原