口腔癌中一氧化氮合酶表达与微血管形成关系的研究
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摘要
一氧化氮（NO）是生物体内一种结构简单的自由基，又是极其重要的
信号分子，参与多种生理过程和包括肿瘤在内的多种病理过程。内源性NO
是由一氧化氮合酶（NOS）催化下，在左旋精氨酸转化为左旋瓜氨酸的过程
中产生。NOS有三种异构酶：神经型NOS（nNOS）、内皮型NOS（eNOS）
和诱导型NOS（iNOS）。现已证实NOS存在于多种肿瘤组织中，如肝癌、
头颈部鳞癌、乳腺癌。有文献报道在一些肿瘤中NOS表达水平与预后相关。
肿瘤组织存在微血管形成的现象，肿瘤的微血管形成是肿瘤发展的必经阶
段，与肿瘤的生长、局部浸润和远处转移相关，而血管内皮生长因子（VEGF）
被认为是最重要的血管形成促进因子。
本课题研究目的：a．研究三种NOS在口腔癌组织中的表达；b．研究
VEGF在口腔癌组织中的表达；c．计数口腔癌组织微血管密度（MVD）值，
研究口腔癌组织微血管形成；d．研究NOS表达水平和口腔癌区域淋巴转移的
关系；e．研究口腔癌NOS表达和VEGF表达、口腔癌组织做血管密度之间
的关系。
浙江大学硕士学位论文
材料与方法
1 选取临床资料完整的口腔癌病例41例作为研究对象，收集年龄、性
别、部位、病理分级、淋巴转移以及临床分期等资料；收集9例正常口腔粘
膜作研究对照。
2 采用兔疫组化SP法检测41 例口腔癌石蜡切片中三种NOS 以及
VEGF的表达；测定口腔癌组织MVD（CD34标记）；检测9例正常口腔粘
膜石蜡切片中三种NOS的表达。
3 统计分析口腔癌NOS表达水平与淋巴转移、病理分级、临床分期的
关系；分析口腔癌NOS表达水平与口腔癌组织微血管密度、VEGF表达水平
之间的夫系。
结果
1 口腔癌 eNOS阳性表达率为 48．80％。其中淋巴结阴性组（LN。）和
淋巴结阳性组（LN＋）eNOS阳性率分别为1905％、80刀0％，其差异有高度
统计学意义（X‘－1523，P－0刀00人病理分级高分化组（GI）和中低分化组
（GZ＋G3） eNOS阳性率分别为 5200％、4375％，其差异无统计学意义；临
床分期（I＋11）组和（Ill＋IV）组 eNOS阳性率分别为 25％、64％，差异有统
计学意义（X’一5939，P—0刀15）。
2，口腔癌 nNOS阳性表达率为引．20％。其中 LN－组和 LN＋组 nNOS阳
性率分别为38刀9％、65刀0％，差异无统计学意义；GI组和GZ＋G3组nNOS
阳性率分别为 52。00％、5000％，差异无统计学意义；临床分期（I＋11）组和
（if＋N）组 nNOS阳性表达率分别为 37，50％、60刀0％，差异无统计学意义。
3 口腔癌 iNOS阳性表达率为 6341％。其中 LN。组和 LN＋组 iNOS阳
性率分别为429％、8500％，其差异有高度统计学意义（X乙7840，p－0刀05人
GI组和 GZ＋G3组iNOS阳性率分别为 72刀0％、56．25％，未显示出统计学差
异；临床分期（1＋11）组和U卜W）组wOS阳性表达率分别为43乃％、
2
浙江大学硕士学位论文
76刀0％，差异有统计学意义（X‘－4374，p－0036）。
’ 0例正常口腔粘膜上皮细胞中 ewos、nwOS和 twos表达均为阴性。
5．口腔癌 MVD均值为 39尸07士门、230。LN－组和 LN＋组 MVD均值分别
为33刀4818657、4670019二80，差异有高度统计学意义（t。4．874，P—0000）；
临床分期（I＋11）组和（m＋w）组 MVD均值分别为 34，438iii、530、
43080t9836，差异有统计学意义（＃2566，P—0刀14）。
6、日腔癌 VEGF阳性表达率为 61．000。其中 LN－组和 LN＋组VEGF阳
性率分别为 4286％、8000％，差异有统计学意义（X＊5939，p－0＊15）；
临床分期（I＋11）组和（m＋w）组 VEGF阳性率分别为 37．50％。76．00O，差
异有统计学意义（X‘－6刀77，P－0014）；VEGF阴性表达组和阳性表达组
MVD均值分别为3212518，617、44．560if0046，差异有统计学意义（t—4刀79，
P—0000）。
7．eNOS阴性表达组和阳性表达组的MVD均值分别为34．524f9，704、
45150t10271，差异有高度统计学意义（t＝3．406，P—0002）；nNOS阴性表
达组和阳性表达组MVD均值分别为36 150d门二46、43095LI〕464，差异有
统计学意义（t＝2058，P—0刀46）；iNOS阴性表达组和阳性表达组 MVD均值
分别为29667L6945、45500t8．910，差异有高度统计学意义（FS、913，
P—0刀00 ）o
8．eNOS阴性表达组和阳性表达组VEGF阳性表达率分别为52．38％、
70刀0％，差异无统计学意义（X 2－l．336，P－0．248）；nNOS阴性表达组和阳
性表达组VEGF阳性表达率分别为50刀0％、7143％，差异无统计学意义；
iNOS阴性表达组和阳性表达组VEGF阳性表达率分别为3333％。76．92％，
差异有高度统计学意义（X‘－7596，p＝0006）。
结论
1 与正常口腔粘膜上皮细胞相比，口腔癌细胞中三种 NOS均有高表达；
Nitric oxide (NO) is a kind of simple but important signal molecule functioning as a kind of second messenger and taking part in many physiological and pathological processes including tumor. Endogenous NO production depends on conversion of the amino acid L-arginine to L-citrulline by a family of enzymes named NO synthase (NOS). NOS has three isoforms: endothelial type NOS (eNOS), neuronal type NOS (nNOS) and inducible type NOS (iNOS). Research so far has proved that NOS exists in many kinds of tumor cells including cell of liver cancer, of breast cancer and of squamous cell carcinoma in head and neck. It is also reported that expression of NOS and its production NO have correlation to prognosis in certain malignant tumors. Angiogenesis existing in many tumor tissues and regarded as an inevitable phase in tumor progress is correlative to tumor growth, local invasion and metastasis. Vascular endothelial growth factor (VEGF) is thought to be the most important promoting factor of angiogenesis.
Objectives of this research include: 1. investigating expressions of three isoforms of NOS in oral squamous cell carcinomas (OSCC); 2. investigating expressions of VEGF in OSCC; 3. examining microvessel density (MVD) of tumor tissues and investigating angiogenesis in OSCC; 4. investigating NOS expression's relationship with metastasis in OSCC; and 5. investigating NOS expression's relationships with VEGF expression and MVD in OSCC. Materials and Methods:
1. 41 cases of OSCC (SCC of tongue, of mandibular gingivae and of buccal mucosa) were selected. Information of these cases was collected including age, gender, location, pathological type, metastases and TNM stage. 9 cases of normal oral mucosa were used as controls to detect NOS expression.
2. Using immunohistochemical SP staining, we examined MVD (labeled by CD34) and investigated expressions of three isoforms of NOS and VEGF in paraffin slices of 41 OSCC cases. As controls, expressions of three isoforms of NOS in 9 cases of normal oral mucosa were also detected.
3. Relationships of NOS expression with metastasis, pathological type and TNM stages were analyzed in OSCC, and so were relationship of NOS expression with VEGF expression and MVD. Results:
1. The positive eNOS expression rate of 41 cases of OSCC was 48.80%. There were a significant statistical difference of positive eNOS expression rate between LN- group (19.05% ) and LN+ group (80.00%) (x 2=15.23, P=0.000), no statistical difference between Gl group (52.00%) and G2+G3 group (43.75%), and a statistical difference between TNM stage (I
+ II) group (25.00%) and TNM stage (III+IV) group (64.00%) ( x 2=5.939,
2. The positive nNOS expression rate of 41 cases of OSCC was 51.20%. There were no statistical difference of positive nNOS expression rate between LN- group (38.09%) and LN+ group (65.00%), no statistical difference between Gl group (52.00%) and G2+G3 group (50.00%), and no statistical difference between TNM stage ( I + II ) group (37.50%) and TNM stage (III+IV) group (60.00%).
3. The positive iNOS expression rate of 41 cases of OSCC was 63.41%. There were a significant statistical difference of positive iNOS expression rate between LN- group (42.90%) and LN+ group (85.00%) ( x 2=7.84, P=0.005), no statistical difference between Gl group (72.00%) and G2+G3 group (56.25%), and a statistical difference between TNM stage ( I +11) group (43.75%) and TNM stage (III+IV) group (76.00%) ( x 2=4.374, P=0.036).
4. There was no positive expression of eNOS, nNOS and iNOS in 9 cases of normal oral mucosa.
5. The mean value of 41 cases of OSCC was 39.707?1.230. The mean value of MVD of LN- group was 33.048?.657 as that of LN+ group 46.700?.280, and there was a significant statistical difference between them (/=4.874, P=0.000). The mean value of MVD of TNM stage ( I + II ) group was 34.43 8? 1.530 as that of TNM stage (III+IV) group 33.048?.657, and there was a statistical difference between them (/=2.566, /MX014).
6. The positive VEGF expression rate of 41 cases of OSCC was 61.00%. There
信号分子，参与多种生理过程和包括肿瘤在内的多种病理过程。内源性NO
是由一氧化氮合酶（NOS）催化下，在左旋精氨酸转化为左旋瓜氨酸的过程
中产生。NOS有三种异构酶：神经型NOS（nNOS）、内皮型NOS（eNOS）
和诱导型NOS（iNOS）。现已证实NOS存在于多种肿瘤组织中，如肝癌、
头颈部鳞癌、乳腺癌。有文献报道在一些肿瘤中NOS表达水平与预后相关。
肿瘤组织存在微血管形成的现象，肿瘤的微血管形成是肿瘤发展的必经阶
段，与肿瘤的生长、局部浸润和远处转移相关，而血管内皮生长因子（VEGF）
被认为是最重要的血管形成促进因子。
本课题研究目的：a．研究三种NOS在口腔癌组织中的表达；b．研究
VEGF在口腔癌组织中的表达；c．计数口腔癌组织微血管密度（MVD）值，
研究口腔癌组织微血管形成；d．研究NOS表达水平和口腔癌区域淋巴转移的
关系；e．研究口腔癌NOS表达和VEGF表达、口腔癌组织做血管密度之间
的关系。
浙江大学硕士学位论文
材料与方法
1 选取临床资料完整的口腔癌病例41例作为研究对象，收集年龄、性
别、部位、病理分级、淋巴转移以及临床分期等资料；收集9例正常口腔粘
膜作研究对照。
2 采用兔疫组化SP法检测41 例口腔癌石蜡切片中三种NOS 以及
VEGF的表达；测定口腔癌组织MVD（CD34标记）；检测9例正常口腔粘
膜石蜡切片中三种NOS的表达。
3 统计分析口腔癌NOS表达水平与淋巴转移、病理分级、临床分期的
关系；分析口腔癌NOS表达水平与口腔癌组织微血管密度、VEGF表达水平
之间的夫系。
结果
1 口腔癌 eNOS阳性表达率为 48．80％。其中淋巴结阴性组（LN。）和
淋巴结阳性组（LN＋）eNOS阳性率分别为1905％、80刀0％，其差异有高度
统计学意义（X‘－1523，P－0刀00人病理分级高分化组（GI）和中低分化组
（GZ＋G3） eNOS阳性率分别为 5200％、4375％，其差异无统计学意义；临
床分期（I＋11）组和（Ill＋IV）组 eNOS阳性率分别为 25％、64％，差异有统
计学意义（X’一5939，P—0刀15）。
2，口腔癌 nNOS阳性表达率为引．20％。其中 LN－组和 LN＋组 nNOS阳
性率分别为38刀9％、65刀0％，差异无统计学意义；GI组和GZ＋G3组nNOS
阳性率分别为 52。00％、5000％，差异无统计学意义；临床分期（I＋11）组和
（if＋N）组 nNOS阳性表达率分别为 37，50％、60刀0％，差异无统计学意义。
3 口腔癌 iNOS阳性表达率为 6341％。其中 LN。组和 LN＋组 iNOS阳
性率分别为429％、8500％，其差异有高度统计学意义（X乙7840，p－0刀05人
GI组和 GZ＋G3组iNOS阳性率分别为 72刀0％、56．25％，未显示出统计学差
异；临床分期（1＋11）组和U卜W）组wOS阳性表达率分别为43乃％、
2
浙江大学硕士学位论文
76刀0％，差异有统计学意义（X‘－4374，p－0036）。
’ 0例正常口腔粘膜上皮细胞中 ewos、nwOS和 twos表达均为阴性。
5．口腔癌 MVD均值为 39尸07士门、230。LN－组和 LN＋组 MVD均值分别
为33刀4818657、4670019二80，差异有高度统计学意义（t。4．874，P—0000）；
临床分期（I＋11）组和（m＋w）组 MVD均值分别为 34，438iii、530、
43080t9836，差异有统计学意义（＃2566，P—0刀14）。
6、日腔癌 VEGF阳性表达率为 61．000。其中 LN－组和 LN＋组VEGF阳
性率分别为 4286％、8000％，差异有统计学意义（X＊5939，p－0＊15）；
临床分期（I＋11）组和（m＋w）组 VEGF阳性率分别为 37．50％。76．00O，差
异有统计学意义（X‘－6刀77，P－0014）；VEGF阴性表达组和阳性表达组
MVD均值分别为3212518，617、44．560if0046，差异有统计学意义（t—4刀79，
P—0000）。
7．eNOS阴性表达组和阳性表达组的MVD均值分别为34．524f9，704、
45150t10271，差异有高度统计学意义（t＝3．406，P—0002）；nNOS阴性表
达组和阳性表达组MVD均值分别为36 150d门二46、43095LI〕464，差异有
统计学意义（t＝2058，P—0刀46）；iNOS阴性表达组和阳性表达组 MVD均值
分别为29667L6945、45500t8．910，差异有高度统计学意义（FS、913，
P—0刀00 ）o
8．eNOS阴性表达组和阳性表达组VEGF阳性表达率分别为52．38％、
70刀0％，差异无统计学意义（X 2－l．336，P－0．248）；nNOS阴性表达组和阳
性表达组VEGF阳性表达率分别为50刀0％、7143％，差异无统计学意义；
iNOS阴性表达组和阳性表达组VEGF阳性表达率分别为3333％。76．92％，
差异有高度统计学意义（X‘－7596，p＝0006）。
结论
1 与正常口腔粘膜上皮细胞相比，口腔癌细胞中三种 NOS均有高表达；
Nitric oxide (NO) is a kind of simple but important signal molecule functioning as a kind of second messenger and taking part in many physiological and pathological processes including tumor. Endogenous NO production depends on conversion of the amino acid L-arginine to L-citrulline by a family of enzymes named NO synthase (NOS). NOS has three isoforms: endothelial type NOS (eNOS), neuronal type NOS (nNOS) and inducible type NOS (iNOS). Research so far has proved that NOS exists in many kinds of tumor cells including cell of liver cancer, of breast cancer and of squamous cell carcinoma in head and neck. It is also reported that expression of NOS and its production NO have correlation to prognosis in certain malignant tumors. Angiogenesis existing in many tumor tissues and regarded as an inevitable phase in tumor progress is correlative to tumor growth, local invasion and metastasis. Vascular endothelial growth factor (VEGF) is thought to be the most important promoting factor of angiogenesis.
Objectives of this research include: 1. investigating expressions of three isoforms of NOS in oral squamous cell carcinomas (OSCC); 2. investigating expressions of VEGF in OSCC; 3. examining microvessel density (MVD) of tumor tissues and investigating angiogenesis in OSCC; 4. investigating NOS expression's relationship with metastasis in OSCC; and 5. investigating NOS expression's relationships with VEGF expression and MVD in OSCC. Materials and Methods:
1. 41 cases of OSCC (SCC of tongue, of mandibular gingivae and of buccal mucosa) were selected. Information of these cases was collected including age, gender, location, pathological type, metastases and TNM stage. 9 cases of normal oral mucosa were used as controls to detect NOS expression.
2. Using immunohistochemical SP staining, we examined MVD (labeled by CD34) and investigated expressions of three isoforms of NOS and VEGF in paraffin slices of 41 OSCC cases. As controls, expressions of three isoforms of NOS in 9 cases of normal oral mucosa were also detected.
3. Relationships of NOS expression with metastasis, pathological type and TNM stages were analyzed in OSCC, and so were relationship of NOS expression with VEGF expression and MVD. Results:
1. The positive eNOS expression rate of 41 cases of OSCC was 48.80%. There were a significant statistical difference of positive eNOS expression rate between LN- group (19.05% ) and LN+ group (80.00%) (x 2=15.23, P=0.000), no statistical difference between Gl group (52.00%) and G2+G3 group (43.75%), and a statistical difference between TNM stage (I
+ II) group (25.00%) and TNM stage (III+IV) group (64.00%) ( x 2=5.939,
2. The positive nNOS expression rate of 41 cases of OSCC was 51.20%. There were no statistical difference of positive nNOS expression rate between LN- group (38.09%) and LN+ group (65.00%), no statistical difference between Gl group (52.00%) and G2+G3 group (50.00%), and no statistical difference between TNM stage ( I + II ) group (37.50%) and TNM stage (III+IV) group (60.00%).
3. The positive iNOS expression rate of 41 cases of OSCC was 63.41%. There were a significant statistical difference of positive iNOS expression rate between LN- group (42.90%) and LN+ group (85.00%) ( x 2=7.84, P=0.005), no statistical difference between Gl group (72.00%) and G2+G3 group (56.25%), and a statistical difference between TNM stage ( I +11) group (43.75%) and TNM stage (III+IV) group (76.00%) ( x 2=4.374, P=0.036).
4. There was no positive expression of eNOS, nNOS and iNOS in 9 cases of normal oral mucosa.
5. The mean value of 41 cases of OSCC was 39.707?1.230. The mean value of MVD of LN- group was 33.048?.657 as that of LN+ group 46.700?.280, and there was a significant statistical difference between them (/=4.874, P=0.000). The mean value of MVD of TNM stage ( I + II ) group was 34.43 8? 1.530 as that of TNM stage (III+IV) group 33.048?.657, and there was a statistical difference between them (/=2.566, /MX014).
6. The positive VEGF expression rate of 41 cases of OSCC was 61.00%. There
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20.Guidi AJ, Abu JG, Berse B, et al. Vascular permeability factor expression and angiogenesis in cervical neoplasia.J Nat Cancer Inst, 1995, 87(16):1237-1245.
21.Tanigawa N, Amaya H, Matsumura M, et al. Extent of tumor vascularization correlates with prognosis and hematogenous metastasis in gatric carcinomas.
Cancer Res，1996，56(11) ：2671-2676．
22． Weidner N，Semple JP,Welch WR，et al．Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma．New Engl J Med，1991， 324(1) ：1-8．
23． Carmeliet P，Jain RK．Angiogenesis in cancer and other diseases．Nature， 2000，407(6801) ：249-257．
24． Ferrara N．Role of VEGF in the regulation ofangiogenesis．Kidney Int，1999， 56(3) ：794-814．
25． Neufeld G，Cohen T，Gengrinovitch S，et al．Vascular endothelial growth factor and its receptors．FASEB J，1999，13(1) ：9-22．
26． Gallo O，Emanuela M，Morbidelli，et al．Role of nitric oxide in angiogenesis and tumor progression in head and neck Cancer．J Natl Cancer Lust，1998， 90(8) ：587-596．
27． Vakkala M，Kahlos K，Lakari E，et al．Inducible nitric oxide synthase expression，apoptosis，and angiogenesis in in situ and invasive breast carcinomas．Clin Cancer Res 2000，6(6) ：2408-2416．
28． Jadeski LC，Lala PK． Nitric oxide synthase inhibition by N(G)-nitro-L-arginine methyl ester inhibits tumor-induced angiogenesis in mammary tumors．Am J Pathol 1999，155(4) ：1381-1390．
29． Campbell SC．Advances in angiogenesis research：relevance to urological ontology．J Urology,1997，158(5) ：1663-1674．
30． Ziche M，Morbidelli L，Choudhuri R，et al．Nitric oxide synthase lies downstream from vascular endothedial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis．J Clin Invest，1997，99(11) ： 2625-2634．
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18.Shpitzer T, Chaimoff M, Gal R Tumor angiogenesis as a prognostic factor in early oral tongue cancer. Arch-Otolaryngol-Head-Neck-Surg, 1996, 122(8):865-868.
19.Mortensen K, Holck S, Christensen IJ,et al. Endothelial cell nitric oxide synthase in peritumoral microvessels is a favorable prognostic indicator in premenopausal breast cancer patients. Clin-Cancer-Res.1999, 5(5):1093-1097.
20.Guidi AJ, Abu JG, Berse B, et al. Vascular permeability factor expression and angiogenesis in cervical neoplasia.J Nat Cancer Inst, 1995, 87(16):1237-1245.
21.Tanigawa N, Amaya H, Matsumura M, et al. Extent of tumor vascularization correlates with prognosis and hematogenous metastasis in gatric carcinomas.
Cancer Res，1996，56(11) ：2671-2676．
22． Weidner N，Semple JP,Welch WR，et al．Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma．New Engl J Med，1991， 324(1) ：1-8．
23． Carmeliet P，Jain RK．Angiogenesis in cancer and other diseases．Nature， 2000，407(6801) ：249-257．
24． Ferrara N．Role of VEGF in the regulation ofangiogenesis．Kidney Int，1999， 56(3) ：794-814．
25． Neufeld G，Cohen T，Gengrinovitch S，et al．Vascular endothelial growth factor and its receptors．FASEB J，1999，13(1) ：9-22．
26． Gallo O，Emanuela M，Morbidelli，et al．Role of nitric oxide in angiogenesis and tumor progression in head and neck Cancer．J Natl Cancer Lust，1998， 90(8) ：587-596．
27． Vakkala M，Kahlos K，Lakari E，et al．Inducible nitric oxide synthase expression，apoptosis，and angiogenesis in in situ and invasive breast carcinomas．Clin Cancer Res 2000，6(6) ：2408-2416．
28． Jadeski LC，Lala PK． Nitric oxide synthase inhibition by N(G)-nitro-L-arginine methyl ester inhibits tumor-induced angiogenesis in mammary tumors．Am J Pathol 1999，155(4) ：1381-1390．
29． Campbell SC．Advances in angiogenesis research：relevance to urological ontology．J Urology,1997，158(5) ：1663-1674．
30． Ziche M，Morbidelli L，Choudhuri R，et al．Nitric oxide synthase lies downstream from vascular endothedial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis．J Clin Invest，1997，99(11) ： 2625-2634．
31． Radisavljevic Z，Avraham H，Avraham S．Vascular endothelial growth factor
up-regulates ICAM-1 expression via the phosphatidylinositol 3 OH-kinase/AKT/Nitric oxide pathway and modulates migration of brain microvascular endothelial cells. J-Biol-Chem, 2000, 275(27) : 20770-20774．
32． Duenas Gonzalez A, Isales CM, del-Mar-Abad-Hernandez-M. Expression of inducible nitric oxide synthase in breast cancer correlates with metastatic disease. Mod-Pathol, 1997, 10(7) : 645-649．
33． Doi C, Noguchi Y, Marat D. Expression of nitric oxide synthase in gastric cancer. Cancer-Lett, 1999, 144(2) : 161-167．
34． Chiarugi V, Magnelli L, Chiarugi A, et al. Hypoxia induces pivotal tumor angiogenesis control factors including p53, vascular endothelial growth factor and the NF кв-dependent inducible nitric oxide synthase and cyclooxygenase-2． J Cancer Res Clin Oncol, 1999, 125(8-9) : 525-528．