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标题:[求助]请教树突状细胞的培养经验

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Table 3. Rate of Breast Cancer per 1,000 Examinations and Distribution of Cancers by Stage, Size, Grade, and Estrogen Receptor
Status Among Postmenopausal Women With Screening Examinations From 1996 to 2000 by HT Use
Hormone Use*
No HT Estrogen Only Estrogen and Progestin
Rate
% of
Cancers Rate
% of
Cancers Rate
% of
Cancers
Stage†
0 0.79 20.8 0.77 19.4 1.11 20.6
I 1.97 51.5 2.21 54.9 2.90 54.3
II 0.91 23.8 0.85 21.6 1.18 21.7
III or IV 0.15 3.9 0.17 4.1 0.19 3.5
Tumor size†‡
10 mm 1.14 36.8 1.19 34.6 1.61 36.9
11-20 mm 1.24 39.7 1.49 43.6 1.90 43.8
20 mm 0.74 23.5 0.71 21.9 0.84 19.4
Tumor grade†‡
1 0.68 24.8 0.78 25.5 1.25 31.9
2 1.20 43.5 1.31 44.2 1.57 41.1
3 or 4 0.87 31.8 0.88 30.2 1.02 27.1
Estrogen receptor
status†‡
Positive 1.96 83.1 2.32 84.2 2.96 89.3
Negative 0.40 16.9 0.38 15.8 0.36 10.7
Abbreviation: HT, hormone therapy.
*Estrogen only group: HT users without a uterus. Estrogen and progestin group: HT users with a uterus.
†Adjusted for age, family history of breast cancer, examination year, time between mammography examinations, and mammography
registry using Poisson regression for rates and logistic/polytomous regression for percentages, standardized to the entire population. Missing
data for non-HT users, HT users without a uterus, and HT users with a uterus: 12%, 13%, and 10% for stage; 12%, 13%, and 13% for tumor
size; 22%, 22%, 20% for tumor grade; and 33%, 28%, and 31% for estrogen receptor status, respectively.
‡Invasive cancer only.
Table 4. Rate of Cancer and 95% CI Among Postmenopausal Women Who Underwent Screening Mammography From 1996 to 2000 by Duration of HT Use
No HT
Duration of Use*
Estrogen Only Estrogen and Progestin
5 Years 5 Years 5 Years 5 Years
No. of examinations 382,435 14,815 67,911 24,819 47,979
No. of cancers 1,803 51 272 81 298
Cancers per 1,000 exams 4.3 3.7 4.0 3.7 6.5
95% CI† 4.2 to 4.5 3.1 to 4.5 3.7 to 4.3 3.2 to 4.3 6.0 to 7.0
Relative risk Referent 0.86 0.92 0.85 1.49
95% CI† 0.71 to 1.03 0.84 to 1.00 0.73 to 0.98 1.36 to 1.63
Abbreviation: HT, hormone therapy.
*Estrogen only group: HT users without a uterus. Estrogen and progestin group: HT users with a uterus.
†Adjusted for age, family history of breast cancer, examination year, time between mammography examinations, and mammography registry using Poisson regression
and standardized to the total population.
4317 BREAST CANCER AND POSTMENOPAUSAL HT
Rate of False-Negative and True-Positive Results per
1,000 Examinations

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The rate of false-negative examination results increased from
0.77 (95% CI, 0.72 to 0.82) in non-HT users, to 0.83 (95% CI,
0.71 to 0.97) in women using estrogen 5 years, to 1.71 (95%
CI, 1.50 to 1.97) in women using estrogen and progestin 5
years (Fig 1). The rate of false-negative examination results with
stage II or higher disease increased from 0.30 (95% CI, 0.27 to
0.32) in nonusers, to 0.38 (95% CI, 0.33 to 0.46) in women using
estrogen, to 0.74 (95% CI, 0.64 to 0.88) in women using estrogen
and progestin (Fig 1).
The rate of true-positive examination results increased from
3.6 (95% CI, 3.4 to 3.7) in nonusers and 3.3 (95% CI, 3.0 to 3.7)
in women using estrogen 5 years, to 5.0 (95% CI, 4.5 to 5.5)
in women using estrogen and progestin 5 years (Fig 1). The
rate of true-positive examination results with stage II or
higher disease increased from 0.77 (95% CI, 0.72 to 0.82) in
nonusers and 0.75 (95% CI, 0.63 to 0.9) in women using
estrogen, to 1.07 (95% CI, 0.89 to 1.29) in women using
estrogen and progestin (Fig 1).
DISCUSSION
We determined the risk of breast cancer and compared the
tumor characteristics among current HT users with those of
nonusers undergoing screening mammography. The likelihood
of being diagnosed with breast cancer was increased 46% among
current estrogen and progestin users who had used HT for 5
years compared with nonusers, but not among estrogen and
progestin users who had used HT for less than 5 years, or
estrogen only users irrespective of duration of use. This supports
previous evidence demonstrating an increased risk of breast
cancer among postmenopausal women who use estrogen and
progestin hormone therapies for a long duration.3-6,25 The risk of
breast cancer was increased both for tumors with favorable and
unfavorable prognostic characteristics with the excess risk somewhat
greater for early-stage, small, low-grade, ER-positive
tumors than for tumors with a higher stage and grade. Estrogenonly
users were slightly more likely to have ER-positive breast
cancer compared with nonusers, but overall risk of breast cancer
was not increased compared with nonusers.
Current HT use has been reported to increase a woman’s RR
of breast cancer by 2% to 3% per year.2,5-7 Some studies have
reported that estrogen plus progestin regimens may be associated
with a greater risk of breast cancer than estrogen-only regimens,
4-6,28 while other studies report that the increased risk with
estrogen is similar to estrogen plus progestin.2,7 It has been
reported that HT users tend to have more in situ or localized
tumors at detection, possibly because of earlier detection by
mammography.2,29 On the other hand, studies have reported that
the extent of disease among HT users is the same as non-HT
Table 5. Relative Risk of Breast Cancer for a Given Tumor Characteristic (type, stage, size, grade, and estrogen receptor status) Among Postmenopausal Women
Using Hormone Therapy Relative to Nonusers
Tumor Characteristic

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Estrogen and Progestin Users for 5
Years Versus Nonusers*
Estrogen and Progestin Users for 5
Years Versus Nonusers† Estrogen-Only Users Versus Nonusers‡
RR 95% CI RR 95% CI RR 95% CI
More favorable prognosis
DCIS 1.41 1.24 to 1.60 0.77 0.62 to 0.96 0.98 0.89 to 1.07
Stage 0 or I 1.51 1.37 to 1.66 0.99 0.84 to 1.16 1.07 1.00 to 1.15
Tumor size 20 mm§ 1.59 1.43 to 1.76 0.95 0.80 to 1.13 1.11 1.03 to 1.19
Grade 1 or 2§ 1.60 1.44 to 1.77 0.85 0.71 to 1.02 1.07 0.99 to 1.15
Estrogen receptor-positive§ 1.72 1.55 to 1.90 1.03 0.87 to 1.22 1.14 1.06 to 1.23
Less favorable prognosis
Invasive 1.51 1.38 to 1.66 0.86 0.74 to 1.01 1.05 0.98 to 1.12
Stage II, III, or IV 1.46 1.30 to 1.63 0.65 0.52 to 0.80 0.91 0.84 to 1.00
Tumor size 20 mm§ 1.24 1.09 to 1.42 0.82 0.66 to 1.01 0.91 0.83 to 1.00
Grade 3 or 4§ 1.54 1.37 to 1.73 0.50 0.39 to 0.64 0.98 0.90 to 1.07
Estrogen receptor-negative§ 0.89 0.77 to 1.03 0.71 0.58 to 0.87 0.94 0.86 to 1.04
NOTE. Relative risk (RR) and 95% CI are adjusted for age, family history of breast cancer, examination year, time between mammography examinations, and
mammography registry using Poisson regression, standardized to the total population.
Abbreviation: DCIS, ductal carcinoma in situ.
*n 298 cancers (248 invasive, 50 DCIS) among 47,979 screening examinations; estrogen and progestin group hormone therapy users with a uterus.
†n 81 (68 invasive, 13 DCIS) among 24,819 screening examinations; estrogen and progestin group hormone therapy users with a uterus.
‡n 1,803 (1,483 invasive, 320 DCIS) among 382,435 screening examinations; estrogen only group hormone therapy users without a uterus.
§Invasive cancer only.
Fig 1. White bars indicate a false-negative rate; vertical striped bars indicate
a false-negative rate associated with stage II, III, or IV disease; solid bars indicate
a true-positive rate; and vertical striped bars indicate a true-positive rate associated
with stage II, III, or IV disease. HT, hormone therapy.
4318 KERLIKOWSKE ET AL
users30 or possibly greater, with more stage II or higher disease
cases, and high S phase fraction tumors.19,20 Also, it has been
reported among current or recent users of HT that increased
duration of use may increase the risk of disease spread.2 In the
largest study to date with 3,202 breast cancer cases, we have
shown that after taking into account factors that enhance the
chance of detecting a tumor with good prognostic features, such
as older age31 and routine screening, women using estrogen and
progestin for 5 years are at increased risk of early stage tumors
with a more favorable prognosis. Importantly, we also found that
women using estrogen and progestin are at increased risk of
tumors, with a less favorable prognosis consistent with the
Women’s Health Initiative randomized controlled trial results.25
One explanation of why HT users have tumors with more
favorable prognosis is that current or recent use of HT promotes
growth of pre-existing, clinically latent, hormone-dependent
cancers of low malignant potential, that may not otherwise
become clinically apparent. In support of this hypothesis are the
findings reported by Beral et al2 that risk of breast cancer

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decreases as time since last HT use increases, such that past users
who have not had HT in more than 5 years are not at increased
risk of breast cancer, regardless of prior duration of use. In
addition, recent oral contraceptive use has been associated with
increased detection of localized tumors that does not persist 10
years or more after cessation of use.32 Other evidence in support
of current or recent HT use acting as a cancer promoter are the
findings that well-differentiated invasive tumors with favorable
histology (papillary, tubular, mucinous, medullary) have been
reported to be more prevalent among HT users than nonusers,33
and that HT users are diagnosed at a younger age compared with
nonusers.15 Our findings that estrogen and progestin users were
younger at diagnosis and more likely to have ER-positive tumors
of smaller size and lower grade compared with nonusers supports
the hypothesis that HT promotes growth of preexisting
clinically latent cancers.
Why would women taking HT undergoing routine screening
mammography be at increased risk of breast cancer with less
favorable prognostic characteristics? Taking HT for more than a
year has been shown to increase mammographic breast density
in approximately 16% to 20% of women,34,35 with greater
increases in mammographic density associated with estrogen and
progestin than with estrogen alone.34,36,37 Increased mammographic
density among women taking HT has been associated
with decreases in the sensitivity and specificity of mammography38-
40 and increases in the minimal detectable size of tumor.41
Consistent with these reports, we found that women using
estrogen and progestin for 5 years have a higher rate of
false-negative examination results, and that these false-negative
examination results are associated with a higher rate of stage II
or higher disease as compared with nonusers. Thus, the
increase in less favorable tumors among long-term estrogen
and progestin users may be due, in part, to tumors obscured by
mammographically dense breasts, that progress between
screening examinations. The higher rate of true-positive
examination results associated with stage II or higher disease
in long-term estrogen and progestin users as compared with
nonusers, suggests that estrogen and progestin also may act
synergistically to promote tumorigenesis and more rapid
tumor growth than estrogen alone.42,43
Most studies have found no significant differences in the ER
profiles of breast cancer in HT users and nonusers.8, 10-15, 18-20,
23,25 One small study reported current HT users are more likely
to be diagnosed with ER positive tumors.9 Another study
reported that only long-term HT use of 57 months or more is
associated with having an ER-positive tumor,4 while one study
reported that estrogen and progestin use is associated with

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having an ER-positive tumor, but that estrogen therapy is not.24
Given that estrogen causes proliferation of ER-positive breast
cells in vitro and in vivo,44,45 it is not surprising that we found
that women using estrogen and progestin and estrogen only were
more likely to be diagnosed with an ER-positive tumor. We
evaluated a large sample of women recently diagnosed with
breast cancer from 1996 to 2001. During that period, ER status
would have been measured with current, new immunohistochemical
assays that minimize misclassification. Our large sample
size and recent period of evaluation with improved ER
detection methods could account, in part, for our ability to detect
a difference in ER status among HT users and nonusers.
We studied a large number of women with breast cancer with
extensive information on tumor characteristics for these women.
Our ability to control for screening interval (surveillance bias) is
another strength of this study. The accuracy of our data depends
on completeness of cancer reporting to the SEER program, state
tumor registries, and pathology laboratories at the mammography
registries, which has been estimated to be more than 94.3%
complete.46 In addition, the cancer rates reported are within the
range of those reported in the literature, for which follow-up has
been reported to be 99.6%.47,48 Tumor size, stage, grade, and ER
status were missing for between 12% to 33% of tumors, due in
part to a change in coding of tumor size and stage by SEER
programs between July 1998 and 1999, which resulted in some
invasive cancers with an in situ component to be coded with an
unknown size. We are not aware of a tumor-reporting bias to
cancer registries related to HT status or history of hysterectomy.
Our finding of similar proportions of missing tumor characteristic
parameters among the three study groups does not support
a tumor-reporting bias. We inferred that women on HT with a
uterus were taking estrogen and progestin, and that women
without a uterus were taking estrogen only, as consistent with
recommended clinical guidelines49 and detailed information
from a subset of mammography registries in this study. Any
misclassification according to type of regimen would make it
more difficult to find an association between HT use and tumor
characteristics and, thus may have attenuated our findings. In
addition, our finding of enhanced risk of breast cancer among
estrogen and progestin users compared to estrogen only users is
consistent with other reports.4-6 We collected information on HT
use at the time of mammography, lessening the possibility of
recall bias. Information on hormone use was self-reported,
perhaps leading to some misclassification, but this is likely to
have been random and to lead to an underestimation of the
association between HT use and tumor characteristics. We were
4319 BREAST CANCER AND POSTMENOPAUSAL HT
not able to determine if tumor characteristics vary by dose or
specific HT regimens.
Millions of women either consider using or begin HT in the
United States each year. Although most use HT for short-term
symptom management, some women may choose to stay on HT
for longer periods. Postmenopausal women with a uterus who
are considering whether to take or stay on HT should be
informed that: (1) using estrogen and progestin HT for 5
years increases the likelihood of developing breast cancer; (2)
estrogen and progestin and estrogen-only hormone therapies
increase the risk of an ER-positive tumor with a higher risk
associated with combination HT regimens; and (3) cancers
associated with estrogen and progestin hormone therapies
include both tumors with favorable prognostic features and
unfavorable prognostic features.

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AUTHORS’ DISCLOSURES OF POTENTIAL
CONFLICTS OF INTEREST
The authors indicated no potential conflicts of interest.
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4320 KERLIKOWSKE ET AL
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发表于 2012-3-18 12:17 资料个人空间短消息加为好友

小中大

自肝癌患者外周血中分离并纯化外周血单个核细胞(PBMC)、T淋巴细胞［3］. 主要步骤如下：以淋巴细胞分离液分离外周血单个核细胞(PBMC)；聚丙烯Petri培养皿培养选择性地除去巨噬细胞；尼龙毛柱过滤除去B细胞；与L-亮氨酸-L-亮氨酰甲基酯共培养选择性除去其中的巨噬细胞、NK细胞及细胞毒性T细胞，余下细胞则主要为T淋巴细胞. 肝癌TAA获取［1，4］：常规培养并收集HepG2肿瘤细胞；低渗冻融HepG2肿瘤细胞，高速离心(10000rpm)并收集HepG2肿瘤细胞碎片作为TAA. TAA刺激DC，制备TAA特异性的DC［1］：TAA(源于1×106 HepG2肿瘤细胞)与PBMC(1×106，含DC)于GM-CSF(

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发表于 2012-3-18 12:17 资料个人空间短消息加为好友

小中大

Sigma 公司的 metrizamide可用来分离树突状细胞，依据细胞的不同密度可将树突状细胞直接分离出来。若想获得较大量的细胞则可从骨髓或脾脏单个核细胞经细胞因子诱导而来。

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